Her2/neu target antigen and use of same to stimulate an immune response

ABSTRACT

A recombinant polynucleotide encoding a Her2/neu target antigen is provided, as is a recombinant Her2/neu target antigen polypeptide. Also provided are methods of using such a recombinant polynucleotide to express a Her2/neu target antigen in a cell. In addition, methods are provided for using the recombinant polynucleotide or the recombinant polypeptide to stimulate an immune response in a subject against cancer that expresses Her2/neu. Methods of making a target antigen also are provided.

FIELD OF THE INVENTION

[0001] The invention relates generally to a tumor vaccine and a methodof stimulating an immune response against a tumor, and more specificallyto an antigenic peptide encoded by a recombinant Her2/neu polynucleotidesequence, to a genetic vaccine containing the recombinant Her2/neupolynucleotide sequence, to a vector containing such a polynucleotide,and to methods of stimulating an immune response in a human patientagainst a tumor expressing Her2/neu.

BACKGROUND INFORMATION

[0002] Although great advances have been made in the methods used fortreating cancer patients, significant problems remain. In general,cancer is treated using surgery, chemotherapy, radiation therapy, or acombination of these methods Surgical methods, however, can be curativeonly when the cancer detected early and has not metastasized. Similarly,radiotherapy, when used, generally only is effective when a tumor islocalized. In many cases, however, a cancer has metastasized by the timeit has been diagnosed and, therefore, chemotherapy, which provides asystemic treatment, is indicated, sometimes in combination with surgeryor radiotherapy. In most cases, chemotherapy suffers from thedisadvantage that it generally is not specific for the cancer cells, butalso kills rapidly dividing normal cells. In fact, toxicity to normalcells generally limits the dose of chemotherapy that a patient cantolerate. In other cases, a hormonal therapy can provide a more specifictreatment, for example, for treating breast cancer where the breastcancer cells express an estrogen receptor. However, cancer cells oftenbecome resistant to a chemotherapeutic, including hormonal, agent and,therefore, become refractory to the treatment.

[0003] Immunotherapy holds great promise for treating cancer because itcan be effective against disseminated disease and because, in theory, itcan directed only against the cancer cells. Immunotherapy can be activeor passive. For active immunotherapy, a tumor antigen is administered toa patient, resulting in the generation of an immune response against theantigen and against cancer cells expressing the antigen. For passiveimmunotherapy, an antibody against a tumor antigen, for example, israised separate from the patient, and is administered to a patienthaving a cancer that expresses the antigen used to raise the antibody.

[0004] Efforts at active immunotherapy of melanoma, for example, havebeen attempted using crude vaccines composed of “killed” melanoma cellsisolated either from the patient to be treated or from another patient,or of lysates or extracts of such cells. However, the use of crudevaccines for immunotherapy is problematic, in part, because the preciseantigenic composition of such vaccines is largely undefined. It isgenerally believed that more effective immunotherapy requires theidentification and isolation of proteins that are expressed relativelyspecifically by cancer cells, preferably on their surface, but are notexpressed on normal cells. However, such cancer cell specific antigensare rare and have been difficult to identify.

[0005] Many cancers are characterized, in part, by an overexpression ofan otherwise normal protein, and efforts to target these overexpressedproteins using immunotherapeutic methods have been attempted. Forexample, the Her2/neu protein can be overexpressed in breast cancercells, and passive immunotherapy using a monoclonal anti-Her2/neuantibody, Herceptin® antibody, has shown a clinical benefit. However,the use of Herceptin® antibody can result in the development ofventricular dysfunction and congestive heart failure and, therefore,requires careful monitoring of a treated patient. Such methods ofpassive immunotherapy also have inherent problems. For example,administration of an antibody as a passive immunotherapy procedure canresult in an immune response generated by the patient against theadministered antibody and can result in an anaphylactic reaction. Also,an administered antibody has a finite lifetime in a patient and,therefore, must be administered several times over a course oftreatment. As such, active immunotherapy against a protein expressed bya cancer cell would be a preferred method of cancer treatment.Unfortunately, as discussed above, few cancer specific proteins havebeen described. Thus, a need exists to identify tumor antigens that canbe used to stimulate an active immune response by a patient against thecancer, without producing undesirable toxicity to normal cells in thepatient. The present invention satisfies this need and providesadditional advantages.

SUMMARY OF THE INVENTION

[0006] The present invention relates to a recombinant polynucleotideencoding a Her2/neu target antigen that includes at least a firstnucleotide sequence, which encodes a Her2/neu target antigen having anamino acid sequence corresponding to about amino acid residues 634 to683 operatively linked to amino acid residues 1035 to 1225 of SEQ IDNO:1, or to about amino acid residues 635 to 685 operatively linked toamino acid residues 1037 to 1257 of SEQ ID NO:2. The present inventionalso relates to a recombinant polynucleotide encoding a Her2/neu targetantigen that includes at least a first nucleotide sequence, whichencodes a Her2/neu target antigen consisting of an amino acid sequencecorresponding to amino acid residues 606 to 683 operatively linked toamino acid residues 1035 to 1255 of SEQ ID NO:1, or to amino acidresidues 608 to 685 operatively linked to amino acid residues 1037 to1257 of SEQ ID NO:2. Recombinant polynucleotides of the invention areexemplified by a polynucleotide encoding the amino acid sequence setforth in SEQ ID NO:4 or SEQ ID NO:10, and by a polynucleotide having thenucleotide sequence set forth in SEQ ID NO:3 or SEQ ID NO:9.

[0007] A recombinant polynucleotide of the invention can further includea second nucleotide sequence, which is operatively linked to the firstnucleotide sequence encoding the Her2/neu target antigen. The secondnucleotide sequence can include an expression regulatory element, forexample, a transcriptional regulatory element, a translationalregulatory element, or a combination thereof, or can encode aheterologous amino acid sequence. Accordingly, a recombinantpolynucleotide of the invention can encode a fusion protein containing aHer2/neu target antigen and a heterologous amino acid sequence. Theheterologous amino acid sequence can be a peptide or polypeptide, whichcan be useful, for example, as a tag to detect or isolate the fusionprotein containing the Her2/neu target antigen, or as aimmunostimulatory polypeptide to further stimulate an immune responsestimulated by the Her2/neu target antigen. A recombinant polynucleotideof the invention can be contained in a cell, which can be a prokaryoticor eukaryotic cell, including a mammalian cell.

[0008] The present invention also relates to a vector, which contains arecombinant polynucleotide encoding a Her2/neu target antigen, which hasan amino acid sequence corresponding to about amino acids 634 to 683operatively linked to amino acids 1035 to 1255 of SEQ ID NO:1, or toabout amino acids 635 to 685 operatively linked to amino acids 1037 to1257 of SEQ ID NO:2, or which corresponds an amino acid sequenceconsisting of amino acid residues 606 to 683 operatively linked to aminoacid residues 1035 to 1255 of SEQ ID NO: 1, or consisting of amino acidresidues 608 to 685 operatively linked to amino acid residues 1037 to1257 of SEQ ID NO:2. The vector can be an expression vector, whichcontains replication or expression regulatory elements necessary forpropagation of the vector or expression of the encoded Her2/neu targetantigen or both in a prokaryotic or eukaryotic cell, for example, amammalian cell. The vector can be a plasmid vector or a viral vector,including a viral vector plasmid. In one embodiment, the viral vector isan alphavirus vector, for example, a Venezuelan equine encephalitisvirus (VEE) vector. In another embodiment, the viral vector is aretrovirus vector, an adenovirus vector, an adeno-associated virusvector, or a vaccinia virus vector. Also provided is a host cellcontaining a vector of the invention, such a host cell being useful, forexample, for propagating the vector or for expressing an encodedHer2/neu target antigen. Where the vector is a viral vector, the hostcell can be a helper cell, which provides in trans factors necessary forreplication and packaging of viral particles containing the viralvector.

[0009] The present invention further relates to a pharmaceuticalcomposition, which contains a recombinant polynucleotide encoding aHer2/neu target antigen having an amino acid sequence corresponding toamino acids 634 to 683 operatively linked to amino acids 1035 to 1255 ofSEQ ID NO:1, or to amino acids 635 to 685 operatively linked to aminoacids 1037 to 1257 of SEQ ID NO:2, or which contains a recombinantpolynucleotide encoding a Her2/neu target antigen consisting of an aminoacid sequence corresponding to amino acid residues 606 to 683operatively linked to amino acid residues 1035 to 1255 of SEQ ID NO:1,or to amino acid residues 608 to 685 operatively linked to amino acidresidues 1037 to 1057 of SEQ ID NO:2. Such a composition is useful, forexample, as a genetic vaccine. In such a composition, the recombinantpolynucleotide can, but need not, be contained in a vector, which can bean expression vector, including a viral expression vector. In oneembodiment, the encoded Her2/neu target antigen has an amino acidsequence as set forth in SEQ ID NO:4. In another embodiment, the encodedHer2/neu target antigen has an amino acid sequence that corresponds toSEQ ID NO:4, for example, a corresponding amino acid sequence of amammalian Her2/neu homolog such as a human Her2/neu homolog (see SEQ IDNO:10).

[0010] The present invention also relates to a recombinant polypeptide,which includes a Her2/neu target antigen having an amino acid sequencecorresponding to about amino acid residues 634 to 683 operatively linkedto amino acids 1035 to 1255 of SEQ ID NO:1, or to about amino acids 635to 685 operatively linked to amino acids 1037 to 1257 of SEQ ID NO:2, orwhich consisting of an amino acid sequence corresponding to amino acidresidues 606 to 683 operatively linked to amino acid residues 1035 to1255 of SEQ ID NO:1, or to amino acid residues 608 to 685 operativelylinked to amino acid residues 1037 to 1257 of SEQ ID NO:2. Therecombinant polypeptide can have a Her2/neu target antigen amino acidsequence as set forth in SEQ ID NO:4, or an amino acid sequencecorresponding to SEQ ID NO:4, particularly an amino acid sequencederived from a mammalian Her2/neu protein, for example, a Her2/neutarget antigen as set forth in SEQ ID NO:10.

[0011] A recombinant polypeptide of the invention can contain aheterologous amino acid sequence operatively linked to the Her2/neutarget antigen. The heterologous amino acid sequence can be any sequenceother than an amino acid sequence that is contiguous in a wild typeHer2/neu protein to the amino acid sequences that are operatively linkedto form a Her2/neu target antigen of the invention. As such, theheterologous amino acid sequence can be a peptide or polypeptide, forexample, an immunostimulatory polypeptide such as a costimulatory B7molecule, or a cytokine such as an interleukin or an interferon. Alsoprovided is a pharmaceutical composition containing a recombinantpolypeptide of the invention.

[0012] The present invention also relates to a method for expressing aHer2/neu target antigen in a cell. Such a method can be performed, forexample, by contacting the cell with a recombinant polynucleotideencoding a Her2/neu target antigen of the invention, for example, aHer2/neu target antigen having the amino acid sequence set forth in SEQID NO:4 or SEQ ID NO:10, under conditions that allow expression of theHer2/neu target antigen by the cell. The contacting can be performed onany cell in which the Her2/neu target antigen can be expressed,including prokaryotic and eukaryotic cells, for example, a mammaliancell. Furthermore, the contacting can be on a cell in culture, forexample, a cell that has been established for passage in culture or apanel of such cells, or a cell that has been removed from the subjectand is contacted ex vivo; or the contacting can be performed in vivo, inwhich case the recombinant polynucleotide can be administered to thesite of cell or to a site to which the cell can migrate, or can beadministered systemically such that it can circulate to the cells inwhich the Her2/neu target antigen is expressed.

[0013] In a method for expressing a Her2/neu target antigen in a cell,the recombinant polynucleotide can, but need not, be contained in avector. Where the recombinant polynucleotide is contained in vector, thevector can be a plasmid or viral vector, and can be a cloning orexpression vector. In one embodiment, the vector is an expressionvector, and in another embodiment, the vector is a viral expressionvector. In addition, the recombinant polynucleotide can, but need not,be operatively linked to an expression regulatory element, for example,a transcriptional regulatory element such as a tissue specific orinducible transcriptional regulatory element, or a translationalregulatory element such as an internal ribosome binding site.

[0014] A method of expressing a Her2/neu target antigen in a cell canfurther include a step of isolating the Her2/neu target antigen from thecell. The Her2/neu target antigen can be isolated from the cell usingany convenient method, including a method based on the identification orbinding of a polypeptide operatively linked to the Her2/neu targetantigen, for example, a polyhistidine tag peptide, which can be bound bya nickel ion chelate. Accordingly, the present invention provides anisolated Her2/neu target antigen obtained by such a method.

[0015] In addition, a method of expressing a Her2/target antigen in acell can further include a step of isolating the cell expressing theHer2/neu target antigen. Such cell can be isolated, for example, byperforming a limiting dilution preparation of cells contacted with therecombinant polynucleotide, and selecting cells in which the encodedHer2/neu target antigen is expressed. Accordingly, the present inventionprovides an isolated cell, which expresses the Her2/neu target antigen,which is obtained by such a method.

[0016] The present invention also relates to a method of stimulating animmune response in a subject against cancer cells that express Her2/neu.Such a method can be performed, for example, by contacting cells withthe recombinant polynucleotide of the invention under conditions thatallow expression from the recombinant polynucleotide of the Her2/neutarget antigen in the cells and contact of the expressed Her2/neu targetantigen with immunoeffector cells, which are involved in an immuneresponse, in the subject. The recombinant polynucleotide can, but neednot, be contained in a vector, which can be, for example, a viralvector.

[0017] In one embodiment of the method of stimulating an immuneresponse, cells are contacted with the recombinant polynucleotide inculture, to generate cells expressing the Her2/neu target antigen, whichare administered to the subject, thereby stimulating an immune responsein the subject against cancer cells that express Her2/neu. The cellsthat are contacted with the target antigen can, but need not, be antigenpresenting cells (APCs) such as dendritic cells. When the cells are notAPCs, the expressed Her2/neu target antigen generally comprises a fusionprotein that can be secreted from the cell expressing the targetantigen.

[0018] In another embodiment of the method, cells other thanimmunoeffector cells are contacted with the recombinant polynucleotidein culture, to generate cells that express the Her2/neu target antigen,which are further contacted in culture with immunoeffector cells togenerate stimulated immunoeffector cells, for example, APCs that canprocess and present the target antigen to other immunoeffector cells.The stimulated cells, either alone or in combination with the cells thatexpress the Her2/neu target antigen, then are administered to thesubject, thereby stimulating an immune response in the subject againstcancer cells that express Her2/neu. The cells that are contacted withthe recombinant polynucleotide and express the Her2/neu target antigen,or the immunoeffector cells, or both, can be autologous cells, which areobtained from the subject to be treated, or can be allogeneic cells withrespect to the subject, including, for example, cells that are haplotypematched with respect to major histocompatibility loci. In still anotherembodiment, the method of stimulating an immune response is performed bycontacting the subject's cells with the recombinant polynucleotide invivo, wherein the cells, which express the Her2/neu target antigen, canfurther contact immunoeffector cells, thereby stimulating an immuneresponse in the subject against cancer cells expressing Her2/neu.

[0019] The present invention also relates to a method ofimmunostimulating immunoeffector cells against cancer cells expressingHer2/neu by contacting cells involved in the immune response with arecombinant polypeptide of the invention. The immunoeffector cells,which are contacted with the recombinant polypeptide, can be any cellsthat can be immunostimulated due to contact with the recombinantpolypeptide, particularly APCs such as dendritic cells, B cells, and thelike. The contacting can be performed on cells in culture, or can beperformed in vivo by administering the recombinant polypeptide to thesubject. The cancer cells, against which the immune response isstimulated, can be any cancer cells that express Her2/neu, particularlycancer cells that overexpress Her2/neu as compared to the level ofHer2/neu expressed by normal cells of the type from which the cancercells are derived. It should be recognized that a method of theinvention also can be used to stimulate an immune response against anycells that express Her2/neu, and particularly against cells thatoverexpress Her2/neu and are involved in a pathologic condition.

[0020] A method of immunostimulating immunoeffector cells against cancercells expressing Her2/neu can further include a step of isolating theimmunostimulated immunoeffector cells. Accordingly, the presentinvention also provides a method of isolating a Her2/neu target antigenimmunostimulated cell, and provides an isolated immunostimulated cellobtained by such a method, for example, an isolated immunostimulateddendritic cell.

[0021] The present invention also relates to a method of making a targetantigen, which can stimulate an immune response against a proteinexpressed on the surface of a cancer cell. Such a method can beperformed, for example, by selecting a first peptide portion of theprotein, wherein the first peptide portion includes the transmembranedomain and has limited homology to proteins other than the proteinexpressed on the surface of the tumor cell; selecting a second peptideportion of the protein, which includes a portion of the cytoplasmicdomain of the protein and has limited homology to proteins other thanthe protein expressed on the surface of the tumor cell; and operativelylinking the first peptide portion and the second peptide portion,thereby making the target antigen.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIGS. 1A and 1B show the amino acid sequences for the human (h;SEQ ID NO:1) and rat (r; SEQ ID NO:2) Her2/neu proteins. In the ratHer2/neu sequence (SEQ ID NO:2), only those amino acid residues thatdiffer from those in the human sequence (SEQ ID NO:1) are shown. Boldindicates the transmembrane domain. Underlining indicates the sequencesused to prepare a Her2/neu target antigen.

[0023]FIG. 2 compares the percent survival (ordinate) with time aftertumor cell challenge (abscissa) of Fisher 344 rats vaccinated with avector containing a Her2/neu target antigen (pITL-rNeu) and ratsvaccinated with an empty vector (pITL). Vaccinations were administeredevery 3 weeks. Tumor challenge occurred 4 weeks after the lastvaccination. Both lines represent administration of 100 ug ofDNA/injection. Animals were euthanized when tumors reached a maximumdimension of 2.5 cm. Survival differences were statistically significantby log rank analysis, p=0.02.

[0024]FIG. 3 compares the percent survival (ordinate) with time aftertumor cell challenge (abscissa) of control Fisher 344 rats (novaccination, open triangles) and Fisher 344 rats treated with 1×10⁶infectious units (ifu) of irradiated 13762 mammary carcinoma cells(closed squares), 1×10⁷ ifu of VRP-rNeu, intramuscularly (IM; closedtriangles), 1×10⁷ ifu VRP-rNeu, subcutaneously (SC; open squares), or1×10⁷ ifu VRP-HA, SC (open circles; see Example 1).

[0025]FIGS. 4A to 4C show the tumor volumes (mm³; ordinate) ofindividual rats with time after tumorcallenge (abscissa). Rats werevaccinated with 1×10⁷ ifu VRP-rNeu, IM (FIG. 4A); 1×10⁶ ifu VRP-rNeu, SC(FIG. 4B); or 1×10⁵ ifu VRP-rNeu, SC (FIG. 4C).

[0026]FIG. 5 shows the percent survival (ordinate) with time after tumorcell re-challenge of surviving animals (see Example 1). Initialtreatments of surviving animals was 1×10⁷ ifu VRP-rNeu, IM (closedsquare); 1×10⁶ ifu VRP-rNeu, SC (closed triangle); 1×10⁵ ifu VRP-rNeu,SC (open circle); or 1×10⁶ irradiated 13762 cells (diamond). Note:closed triangle and open circle are included within the closed square.

[0027]FIGS. 6A and 6B show nucleotide (SEQ ID NO:3; start to stop codon)and amino acid (SEQ ID NO:4) sequences of a Her2/neu target antigen,which includes two peptide portions of rat Her2/neu (i.e., amino acidresidues 635 to 685 and 1037 to 1257 of SEQ ID NO:2; see, also, EntrezP06494, and SEQ ID NOS:5 and 7), which are operatively linked through aserine residue (single underline) and encoded by a portion of the BspE1restriction endonuclease recognition site (single underline) that wasengineered into the polynucleotides comprising the encoding recombinantpolynucleotide. Double underlining indicates sequence added to initiatetranscription and maintain reading frame. Single underline indicates thefusion site (note: a terminal adenine residue was added to the “Proximalrat Her2/neu nucleotide sequence (SEQ ID NO:6; see, also GenBankNucleotide Acc. No. NM_(—)017003-nucleotides 1928 to 2081) to generate aBspE1 site; underlined). The construction process also added a serine(S) to the amino acid residues 638-688 of the “Proximal rat Her2/neupolypeptide” (SEQ ID NO:5; Entrez P06494, amino acid residues 635 to685; see, also, GenBank Protein Acc. No. NP_(—)058699, amino acidresidues 638 to 688—sequences identical in these regions). The “Terminalrat Her2/neu polypeptide” (SEQ ID NO:7 (amino acids 1037-1257 of EntrezP0649; see also, GenBank Protein Acc. No. NP_(—)058699, amino acidresidues 1040 to 1260—identical sequences in these regions); and the“Terminal rat Her2/neu nucleotide sequence (SEQ ID NO:8; GenBankNucleotide Acc. No. NM_(—)017003, nucleotides 3128 to 3799; note:introduced BspE1 site underlined—“G” residue added to maintain readingframe of fusion target antigen) also are shown.

[0028]FIGS. 7A and 7B show the nucleotide (SEQ ID NO:9) and amino acid(SEQ ID NO:10) sequences for the “HUMAN Her2/neu” target antigen.Underline and double underlining as in FIGS. 6A and 6B. Similarly toFIGS. 6A and 6B, the proximal and terminal Her2/neu sequence used toconstruct the fusion target antigen are shown. “Proximal human Her2/neupolypeptide” (SEQ ID NO:11; Entrez NP_(—)004439 or P04626, amino acidresidues 634 to 683) and “Proximal human Her2/neu nucleotide” (SEQ IDNO:12; GenBank Nucleotide Acc. No. NM_(—)004448, nucleotides 2040 to2199) are shown, as are the “Distal human Her2/neu” polypeptide (SEQ IDNO:13; Entrez NP 004439 or P04626, amino acid residues 1034-1255) and“Distal human Her2/neu nucleotide (SEQ ID NO:14; GenBank Nucleotide Acc.No. NM_(—)004448, nucleotides 3253 to 3918) sequences. In SEQ ID NO:14,“a/g” represents a site specific (a to g) mutation induced to create theBspE1 site for fusion. “G” indicates a guanine residue added to maintainthe reading frame of the fusion target antigen.

DETAILED DESCRIPTION OF THE INVENTION

[0029] The present invention provides compositions useful forstimulating an immune response against cancer cells that express aHer2/neu protein, and also provides methods of using the compositions tostimulate such an immune response. As disclosed herein, the compositionsof the invention include, or encode, a recombinant polypeptide (Her2/neutarget antigen) formed by linking two non-contiguous peptide portions ofHer2/neu such that they are expressed as a single polypeptide. Her2/neuis a member of the epidermal growth factor receptor family of proteinsand is normally expressed in various cell types. In addition, Her2/neuis over-expressed in various types of cancer, including in about 20 to30% of breast cancers, in adenocarcinomas of the ovary, salivary gland,stomach and kidney, in colon cancers, and in non-small cell lung cancer(see, for example, Fendly et al., J. Biol. Resp. Mod. 9:449-455, 1990,which is incorporated herein by reference).

[0030] Since Her2/neu is a “self” protein, it is not normally recognizedby the immune system, and any robust immune response generated against anormal Her2/neu protein is likely to elicit deleterious crossreactiveautoimmune phenomena. As such, efforts have been made to identifypeptide antigens of Her2/neu that can be used to stimulate an immuneresponse against Her2/neu expressing tumor cells in a cancer patient.For use as a cancer vaccine, it is preferable if the target antigenelicits a T helper-1 (Th1) cell response and a cytotoxic T lymphocyte(CTL) response. Such an immune response is generally observed forproteins expressed in the intracellular compartment, and lessprominently with extracellular or secreted proteins. In addition, rapiddegradation of a protein can contribute to establishing such a bias inthe immune response, facilitating antigen presentation and, perhaps,assisting in establishing specificity of the immune response (see, forexample, Montoya and Del Val, J. Immunol. 163:1914-1922, 1999; Yewdellet al., Adv. Immunol. 73:1-77, 1999). Although peptide strategies can beused to obtain such a biased immune response, the occurrence of antigenloss variants (Marincola et al., Adv. Immunol. 74:181-273, 2000; Rikeret al., Surgery 126:112-120, 1999; Slingluff et al., Cancer Immunol.Immunother. 48:661-672, 2000) indicates that broader or poly-epitopeimmune responses will be more efficacious. Accordingly, an effort wasmade to design a Her2/neu target antigen that preferentially stimulatesa broad based Th1 and CTL immune response.

[0031] As disclosed herein, a Her2/neu target antigen was derived byidentifying two peptide portions of the human Her2/neu protein thatdemonstrated the lowest degree of sequence homology with other knownnormal human proteins. These two peptides include one peptideencompassing the transmembrane (TM) domain and a portion of theextracellular domain and a second peptide including the C-terminaldomain (see FIG. 1, underlining). These sequences were combined toencode a single polypeptide that did not show any new homology to normalproteins (see Example 1). As such, a Her2/neu target antigen of theinvention minimizes the likelihood that any potential crossreactiveautoimmune response will occur, while maximizing specificity of theelicited immune response. Furthermore, since the Her2/neu target antigenis constructed predominantly of Her2/neu sequences that areintracellular, and represents a truncated or partial protein sequence,it can be rapidly degraded by intrinsic antigen processing to produce Tcell epitopes that are presented in the context of MHC class I and,therefore, available for CTL recognition.

[0032] The use of a Her2/neu target antigen of the invention, either asa polynucleotide or as a polypeptide, to stimulate an immune responseprovides the advantage that specific peptide T cell epitopes that bindparticular MHC class I alleles need not be defined. As compared to Tcell epitopes, the Her2/neu target antigen of the invention canstimulate a more broad based and less restricted immune response thancan a T cell epitope peptide. In addition, the larger polypeptide allowsfor the intrinsic antigen processing machinery to generate appropriatepeptides for the expressed MHC alleles. To otherwise generate suchpeptides, all of the MHC alleles for a patient would need to be known,and the appropriate peptides would have to be characterized for anysingle protein antigen. The use of a Her2/neu target antigen of theinvention also provides advantages over the use of a full lengthpolypeptide antigen, which can generate a less specific immune response,more crossreactive and potentially deleterious immune reactivity, andless of a Th1 CTL bias.

[0033] Class I MHC molecules are, or can be, expressed by all nucleatedcells and present T cell epitopes to cytotoxic T lymphocytes (CTLs). Ingeneral, the epitopes presented by class I MHC molecules are produced byproteolysis of endogenously expressed proteins, including proteinsexpressed in virally infected cells and in tumor cells. The epitopelikely associates with the class I molecule in the endoplasmicreticulum, then the complex is transported to the cell surface. CTLs,which express the CD8 surface antigen (“CD8⁺”) and a CTL receptor, thenbind the epitope associated with the class I molecule, therebyactivating the effector function of the CTLs (see, generally, Kuby,“Immunology” 3d ed. (W.H. Freeman and Co., 1997).

[0034] Class II MHC molecules, which are expressed by antigen presentingcells, present a T cell epitope to helper T cells (“Th cells”),stimulating the Th cells, such stimulation being effective in immunityto tumors (see Abbas et al., “Cellular and Molecular Immunology,” seconded. (W. B. Saunders Co. 1995); Kuby, supra, 1997). The epitopes that arebound by class II molecules generally are derived by proteolysis ofexogenous proteins, which are internalized in the APC by phagocytosis orendocytosis. In addition, APCs, such as macrophages, can expressco-stimulatory B7 molecules B7-1 (CD80) and B7-2 (CD86), which arerecognized by a cell surface molecule (CD28) that is expressed bycertain T cells, including naive T cells, and is involved in activationof the T cells. Binding of a T cell epitope and B7 molecule by Th cellsstimulates activation of two subsets of Th cells, Th1 cells, whichexpress interleukin-2 (IL-2), interferon-γ, tumor necrosis factor-β andtumor necrosis factor-α and are involved in the cell-mediated immunefunctions, including activation of CTLs, and Th2 cells, which secreteIL-4, IL-5, IL-6 and IL-10 and are involved in the activation of B cells(see Kuby, supra, 1997; chaps. 1, 10 and 12).

[0035] In 2000, over 184,000 new cases of breast cancer will bediagnosed, accompanied by over 41,000 deaths due to this malignancy.Although significant progress has been made in the surgical,radiotherapeutic, and medical treatment of this disease, none of thesemodalities, alone or in combination, provides curative therapy for themajority of patients with advanced breast cancer. Interestingly,patients with significant inflammatory infiltrates in the primary tumor(medullary carcinoma) have significantly improved survival despite ahigher degree of cellular anaplasia. Elution of antigens from immunecomplexes present in sera of patients with breast carcinoma revealsmultiple oncogenic antigens (Croce et al., Cancer Immunol. Immunother.40:132-137, 1995, which is incorporated herein by reference). Breastcancer patients have been reported to have antibodies to mutated p53proteins and Her2/neu (Angelopoulou et al., Clin. Biochem. 33:53-62,2000; Disis et al., Clin. Cancer Res. 6:1347-1350, 2000, each of whichis incorporated herein by reference). Patients with breast cancer canhave a cellular response to autologous tumor associated antigens (TAAs)such as; mutated p53, HER2/neu, the MUC-1 antigen and sialyl-Tn (Disiset al., supra, 2000; Sandmajer et al., J. Immunother. 22:54-66, 1999).These findings, in addition to the association of improved survival withinflammatory infiltration of primary breast tumors, demonstrate thatpatients can mount an immune response to the malignant cells of breasttumors. As such, it is reasonable to hypothesize that a robust,antigen-specific immunotherapeutic strategy for breast cancer can haveclinical efficacy.

[0036] TAAs are generally recognized by the immune system as smallfragments of proteins presented to the immune system in the context ofmajor histocompatibility complex (MHC) molecules on the surface ofantigen presenting cells and tumor cells rather than as large intactproteins. Cytotoxic T lymphocytes (CTL) respond to antigens presented onMHC class I. Helper T lymphocytes respond to antigens presented on MHCclass II. Anti-tumor immune responses can be broadly classified intoTh1-biased and Th2-biased immune responses. Th1-biased responses supportthe CTL component of the cellular immune response, and Th2-biasedresponses support the humoral arm. In selected tumor systems, humoral(antibody) responses have had some clinical utility. In breast cancerpatients, therapeutic administration of an antibody to the Her 2/neumolecule, Herceptin® antibody, has resulted in clinical responses.However, these clinical responses appear to be a result of interferencewith receptor signaling rather than immunologically-mediated cellulardestruction. Although there is a theoretical role for antibodies in theanti-tumor immune response, immunotherapeutic strategies that can elicitTh1-biased immune responses seem most likely to result in theimmunological destruction of tumor cells.

[0037] The selection of a potential target TAA can substantiallyinfluence the type and class of immune response, the specificity, andpotential efficacy of an immunotherapeutic strategy. TAAs containingextracellular components can be targets for humoral immune responses.However, most characterized TAAs are intracellular proteins and areunavailable for reacting with circulating antibodies. Furthermore,intracellular proteins are thought to be presented primarily on MHCclass 1 and elicit CD 8+ T cell responses. In order to obtain optimalspecificity, the selection of a putative target sequence within a TAAshould avoid regions with a high degree of homology to other familymembers or other normal cellular proteins. Additionally, the immunesystem avoids anti-self reactivity by negative selection and theinduction of peripheral tolerance to widely distributed selfantigens/epitopes and this suggests that epitopes from regions of highhomology are less likely to be recognized by the immune system. Thus,selection of intracellular components should bias the elicited responsetoward a CTL/Th 1 immune response and avoidance of these regions of highhomology should enhance the potential for eliciting efficaciousantigen-specific immune responses. The recent recognition of “antigenloss variants” resulting in tumor escape from single antigenimmunotherapeutic strategies (Slingluff et al., supra, 2000; Riker etal., supra, 1999) suggests that the use of polyvalent (multiple antigen)immunotherapies will be necessary for optimal clinical efficacy. Amongcurrently available technologies, genetic or molecular vaccines appearto be the most efficient for generating individualized polyvalent(multiple TAAs) immunotherapeutic strategies.

[0038] Although immune responses to various TAAs have been elicited by anumber of immunotherapeutic modalities the magnitude of these responsesare markedly less than those elicited by pathogenic organisms or inallogeneic graft rejection. Furthermore, using MHC tetramer stainingwith immunodominant peptide antigens in the setting of patients withmelanoma, “antigen educated T lymphocytes” can be identified and havebeen recognized to be functionally anergic, suggesting that the immunesystem is “crippled” with regard to its recognition of TAAs (Valmori etal., Internatl. Immunol. 11:1971-1980, 1999; Lotze, in “KeystoneSymposia on Cellular Immunity and Immunotherapy of Cancer (Santa Fe N.Mex. 2000), each of which is incorporated herein by reference). Thus,new immunotherapeutic approaches will need to include strategies toovercome what amounts to a “high activation barrier” for mounting aneffective anti-tumor immune response.

[0039] One point of attack for enhancing the immune response to aspecific antigen is at the initial point of presentation of antigen tothe immune system. Numerous strategies have been developed foraugmenting the capacity for presentation of TAAs to the immune system.The use of cytokine “biological adjuvants” such as GM-CSF has resultedin augmented immune responses, although improved clinical responses inhumans have not yet been rigorously demonstrated. Another strategy hasused viral vectors containing the TAA and costimulatory molecules, withor without cytokines, in order to make any transfected cell into apotent antigen presenting cell. This strategy has produced an improvedmagnitude of immune responses in animal models. Other strategies havefocused on the most potent antigen presenting cell (APC) within theimmune system in an effort to improve the magnitude of the elicitedimmune response, the dendritic cell (DC).

[0040] DCs can be quite pleiotropic and manifest several phenotypes,including immature and mature phenotypes (Nelson et al., FASEB J.13:2021-2030, 1999, which is incorporated herein by reference). DCsexpress high levels of co-stimulatory and MHC molecules that are furtherup-regulated upon acquisition of the mature phenotype (CD83+ in humans).DCs with the immature phenotype reside in the periphery and are veryactive in antigen uptake and processing. In contrast, DCs with themature phenotype are found almost exclusively in secondary lymphoidorgans, where they have down-regulated their antigen uptake andprocessing pathways while having markedly enhanced theirimmunostimulatory capacity.

[0041] Various strategies have been used for loading DCs with TAAs inthe ex vivo setting. DCs have been loaded with peptides from specificTAAs known to bind particular HLA molecules (Fong and Engleman, Ann.Rev. Immunol. 18:245-273, 2000, which is incorporated herein byreference). However, this strategy is complicated by the recentlydescribed, potentially unfavorable kinetics of peptide degradation byDCs. DCs have been fused with tumor cells and transfected with amplifiedtumor-derived mRNA or cDNA. These latter efforts, although targetingmultiple TAAs, have the disadvantage of lacking the ability to defineand monitor antigen-specific responses because the specific antigens arenot necessarily known. Thus, monitoring of anti-tumor immune responsesmust rely upon the most general measures of immune responses and uponclinical outcome. DCs have also been transfected with viral constructs,with successful expression of the encoded TAA, induction of anantigen-specific immune response and spontaneous acquisition of themature phenotype. However, this strategy and most of the others involveadoptive transfer of ex vivo manipulated DCs of the mature phenotype.Trafficking of adoptively transferred DCs has been problematic withscant evidence that the DCs traffic appropriately to either secondarylymphoid organs or sites of tumor (Barratt-Boyes et al., J. Immunol.164:2487-2495, 2000; Morse et al., Cancer Res. 59:56-58, 1999, each ofwhich is incorporated herein by reference). The capacity to load ortransfect DCs of the immature phenotype with specific TAAs in vivo wouldcircumvent the limitations noted above.

[0042] Venezuelan Equine Encephalitis (VEE) virus and the engineeredalpha virus replicon particles derived from VEE (VRP) have a pronouncedtropism for DCs and lymphocytes in vivo and in vitro (Davis et al., J.Virol. 70:3781-3787, 1996; MacDonald and Johnston, J. Virol. 74:914-922,2000, each of which is incorporated herein by reference). VEE is apositive strand RNA virus that can cause zoonotic human diseasecharacterized by an undifferentiated febrile illness, which can alsoinclude initial lymphopenia. In rodents and large animals, the infectionby VEE can be rapidly fatal and is similarly associated with an earlylymphopenia, which may be mediated by the tropism of VEE for cells ofthe immune system and the resultant cytopathic effect. VEE and VRPtransfect a subset of human DCs of the immature phenotype. The alphavirus replicon vector system derived from VEE constitutes a replicationdeficient viral vector devoid of coding sequences for structural geneswith coding sequences for target antigens (Pushko et al., Virology239:389-401, 1997, which is incorporate herein by reference). The VRPsare constructed by co-electroporation of the VRP RNA and two separatehelper RNAs encoding the required structural genes for permissiveencapsulation of the VRP RNA. The resultant VRPs demonstrate anidentical cellular tropism as VEE in the absence of particularattenuating mutations (Davis et al., Virology 212:102-110, 1995, whichis incorporated herein by reference). The positive strand RNA nature ofthe genome of both VEE and the VRP results in rapid and robust proteinproduction. In the case of the VRP, the target antigen is similarlyexpressed at high levels. Furthermore, at least in the case of aputative retroviral antigen (SIV and HIV-1), the elicited immuneresponse includes both humoral and CTL responses (Caley et al., Vaccine17:3124-3135, 1999; Davis et al., J. Virol. 74:371-378, 2000, each ofwhich is incorporated herein by reference). The use of the VRP vectorsystem allows for in vivo targeting of antigen expression in the mostpotent antigen presenting cells and the potential for augmenting aTh1/CTL-biased immune response to selected TAAs.

[0043] The present invention provides recombinant polynucleotidesencoding a recombinant Her2/neu target antigen. Such recombinantpolynucleotides are useful for genetic vaccination strategies, and therecombinant Her2/neu target antigen polypeptides are useful forpolypeptide vaccination strategies. As used herein, the term “geneticvaccination” refers to the use of a nucleic acid sequence, which encodesan antigen, to stimulate an immune response, wherein expression of theencoded antigen leads to stimulation of the immune response. Incomparison, the term “polypeptide vaccination” is used herein to referto the use of a polypeptide antigen to stimulate an immune response. Asdisclosed herein, a genetic or polypeptide vaccination can be performedby administering a recombinant polynucleotide or polypeptide directly toan individual, or by contacting the recombinant polynucleotide orpolypeptide with cells in culture, which either can be administered tothe subject to treated or can be further contacted with immunoeffectorcells, which are administered to the subject.

[0044] As used herein, the term “stimulate an immune response” or“immunostimulate” refers to an activation of immunoeffector cells asnormally occurs upon contact of such cells with an antigen. As usedherein, the term “immunoeffector cells” refers to cells that aredirectly involved in generating or effecting an immune response,including B lymphocytes (B cells), T cells, including CTLs and Th cells,and antigen presenting cells (APCs) such as dendritic cells, mononuclearphagocytic cells, macrophages, including Langerhans cells and, inhumans, venular endothelial cells (and B cells). Stimulation ofimmunoeffector cells can be identified using well known immunologicmethods, which are selected based on the particular immunoeffector cellsthat are to be stimulated. For example, stimulation of a T cell responsecan be identified by detecting a delayed-type hypersensitivity responsein a subject, which is predictive of T cell immunity due to Her2/neupeptide immunization (see Disis et al., supra, 2000), or by detectingincreased and specific CTL activity using a cell based assay (see Abbaset al., supra, 1995). Immunostimulation of B cells can be identified, ifdesired, by detecting the conversion of a stimulated B cell to a plasmacell, or by detecting expression (or increased expression) of aparticular antibody (see, for example, Harlow and Lane, “Antibodies: Alaboratory manual (Cold Spring Harbor Laboratory Press 1989)).

[0045] A recombinant polynucleotide of the invention encodes a Her2/neutarget antigen, and includes at least a first nucleotide sequence, whichencodes a Her2/neu target antigen having an amino acid sequencecorresponding to about amino acid residues 634 to 683 operatively linkedto amino acid residues 1035 to 1255 of SEQ ID NO:1 (see SEQ ID NOS:9 and10), or to about amino acid residues 635 to 685 operatively linked toamino acid residues 1037 to 1257 of SEQ ID NO:2 (see SEQ ID NOS:3 and4). As used herein, the term “about,” when used in reference to aminoacid residues of a polypeptide such as SEQ ID NO:1, means the specifiedamino acid residues or an amino acid sequence containing one to eightadditional contiguous amino acid residues of the specified sequence, orone to eight fewer amino acid residues than the specified residues. Inaddition, a recombinant polynucleotide of the invention encodes aHer2/neu target antigen, and includes at least a first nucleotidesequence, which encodes a Her2/neu target antigen consisting of an aminoacid sequence corresponding to amino acid residues 606 to 683operatively linked to amino acid residues 1035 to 1255 of SEQ ID NO:1,or corresponding to amino acid residues 608 to 685 operatively linked toamino acid residues 1037 to 1257 of SEQ ID NO:2. As such, therecombinant polynucleotides, and encoded polypeptides, aredistinguishable from previously described Her2/neu target antigens (seeInternatl. Publ. No. WO 98/06863, which is incorporated herein byreference).

[0046] As used herein, the term “corresponding,” when used in referenceto a specified amino acid sequence, means an amino acid sequence of aHer2/neu homolog, which, when aligned with SEQ ID NO:1 (Entrez ProteinAccession No. P04626, which is incorporated herein by reference) or SEQID NO:2 (Entrez Protein Accession No. P06494, which is incorporatedherein by reference; see, also Entrez Protein Accession No. NP004439,which substantially encodes SEQ ID NO:2, including a few additionalamino acid residues before the initial methionine of SEQ ID NO:2, butotherwise is identical), encompasses the same portion of the Her2/neuprotein as the specified amino acids. As shown in FIG. 1, the amino acidsequences of human Her2/neu (SEQ ID NO:1) and rat Her2/neu (SEQ ID NO:2)are aligned, and, for example, amino acid residues 634 to 683 and 1035to 1255 of SEQ ID NO:1 are indicated by underlining. In view of thisdisclosure, it will be recognized that an amino acid sequence of anyother Her2/neu protein, including other mammalian Her2/neu homologs,that correspond to those shown in FIG. 1 similarly can be used toconstruct a Her2/neu target antigen of the invention. In general, a“corresponding” amino acid sequence is at least about 60% identical to areference sequence (e.g., amino acid residues 635 to 685 SEQ ID NO:2),generally at least about 70% identical, and can be 80% identical, or 90%identical, or more. For example, amino acid residues 634 to 683 of SEQID NO:1 (human Her2/neu) are about 72% identical (35/49 residues) to thecorresponding sequence of rat Her2/neu (amino acid residues 635 to 685of SEQ ID NO:2; the exemplified proximal rat Her2/neu target sequencealso contains additional amino acid residue 635 (serine) as compared tothe corresponding exemplified portion of the proximal human Her2/neutarget sequence; see underlining in FIG. 1A; see, also, SEQ ID NO:5). Itshould be recognized that an amino acid residue of a Her2/neu targetantigen can be a modified amino acid, for example, a D-amino acid,which, for purposes of identifying a “corresponding” amino acidsequence, is considered to be identical to the unmodified amino acid(e.g., to an L-amino acid).

[0047] Recombinant polynucleotides of the invention are exemplified bypolynucleotides encoding the amino acid sequences set forth in SEQ IDNO:4 and SEQ ID NO:10, and by polynucleotides having the nucleotidesequences set forth in SEQ ID NO:4 and SEQ ID NO:9, respectively. Asshown in FIGS. 6A and 6B, an exemplified Her2/neu target antigenincludes two peptide portions of rat Her2/neu (i.e., amino acid residues635 to 685 and 1037 to 1257 of SEQ ID NO:2; see SEQ ID NO:4; see, also,SEQ ID NOS:5 and 7), which are operatively linked through a serineresidue (underlined; see, also, SEQ ID NO:4), which is encoded by aportion of the BspE1 restriction endonuclease recognition site (singleunderline; see, also, SEQ ID NO:3) that was engineered into thepolynucleotides comprising the encoding recombinant polynucleotide.Similarly, FIGS. 7A and 7B show corresponding nucleotide (SEQ ID NOS:12and 14) and amino acid sequences (SEQ D NOS:11 and 13) of human Her2/neuuseful for preparing a target antigen of the invention. In addition, theexemplified recombinant Her2/neu target antigens contain additionalN-terminal methionine and alanine residues, which were included in theencoding recombinant polynucleotide to facilitate expression and tomaintain the reading frame (see SEQ ID NOS:4 and 10). As such, it shouldbe recognized that a recombinant Her2/neu target antigen can containamino acid residues in addition to those specified.

[0048] Accordingly, a recombinant polynucleotide of the invention canfurther include, in addition to the Her2/neu target antigen codingsequence, a second nucleotide sequence, which is operatively linked tothe sequence encoding the target antigen. As used herein, the term“operatively linked” means that a first and second (or more) nucleotidesequence are joined together such that each maintains all or a relevantpart of its function. For example, where a first nucleotide sequenceencodes a Her2/neu target antigen and a second nucleotide sequenceincludes a transcriptional promoter, the two sequences, when operativelylinked, function such that the promoter can direct transcription of thecoding sequence such that a transcribed mRNA can be translated.Similarly, when a first and second nucleotide each encode a polypeptide,the two sequences, when operatively linked, can encode all or a portionof each polypeptide, as desired, and can encode a fusion polypeptideincluding the two polypeptides, if desired. The term “operativelylinked” also is used herein to refer to two or more peptides orpolypeptides that are joined together such that they form a singlerecombinant polypeptide or fusion protein. For convenience ofdiscussion, the term “recombinant polypeptide” is used herein to referto a Her2/neu target antigen of the invention, which comprises aproximal and distal portion of a Her2/neu polypeptide, whereas the term“fusion protein” is used to refer to two or more other polypeptidecomponents that are expressed as a single polypeptide or to arecombinant polypeptide of the invention operatively linked to one ormore other peptides or polypeptides.

[0049] A nucleotide sequence operatively linked to the recombinantpolynucleotide encoding the Her2/neu target antigen can include anexpression regulatory element or can encode a heterologous amino acidsequence, for example, a polypeptide. As used herein, the term“expression regulatory element” refers to a nucleotide sequence thatacts in cis to regulate the level of transcription of an operativelylinked polydeoxyribonucleotide or to regulate the level of translationof polyribonucleotide (i.e., a transcriptional or translationalregulatory element, respectively). Thus, an expression regulatorysequence can be a promoter, enhancer, silencer, insulator, transcriptionterminator, start codon (ATG), splicing signal for intron excision andmaintenance of the correct reading frame, STOP codon, ribosome bindingsite such as an internal ribosome entry site, or the like.

[0050] A transcriptional regulatory element can be a constitutivelyactive regulatory element or can be an inducible regulatory element,including an inducible regulatory element that is inactive in theabsence of an inducing agent, or an element that is active at a basallevel and is induced to a higher level in the presence of the inducingagent. In addition, the transcriptional regulatory element can be atissue-specific regulatory element, which is active in only one or a fewspecific cell types, or can be a developmental stage specific regulatoryelement, which is active only during a certain stage of development,including in a pathologic state having the characteristics of aparticular stage of development, for example, a cancer.

[0051] A transcriptional regulatory element can be a viraltranscriptional regulatory element, including, for example, acytomegalovirus promoter, an SV40 promoter or enhancer, a retrovirus U3enhancer such as an RSV U3 enhancer, or the like. A transcriptionalregulatory element also can be a transcriptional regulatory element of aeukaryotic gene, including, for example, a metallothionein promoter,which is constitutively expressed in most cell types and also isinducible; a myoD promoter, which is a tissue specific promoter that isactive in differentiating or differentiated muscle cells; a lckpromoter, which is a tissue specific promoter active in T cells; amyelin basic protein promoter, which is active in myelinating glialcells; a nestin promoter, which is active in neural stem cells; adopamine β-hydroxylase or preproenkephalin promoter, which is active inparticular neuronal cells; or a chemokine promoter such as a fragment ofthe RANTES promoter (Internatl. Publ. No. WO 98/06863). In addition, thetranscriptional regulatory element can be an individual enhancer elementfrom a eukaryotic gene, and can be present as a single copy or asmultiple linked copies, or can be combined with one or more otherelements present as a single copy or as multiple linked copies. Examplesof such individual elements include an AP1 binding site, an Sp1 bindingsite, an NF κB binding site, a serum response factor binding site, ahypoxia inducing factor binding site, a cAMP responsive element, and aphosphoglycerate kinase 1 (PGK1) enhancer.

[0052] As used herein, the term “inducible,” when used in reference to atranscriptional regulatory element, means a nucleotide sequence that,when present in a cell exposed to an inducing agent, effects anincreased level of transcription of an operatively linked expressiblepolynucleotide as compared to the level of transcription, if any, in theabsence of an inducing agent. The term “inducing agent” is used to referto a chemical, biological or physical agent that effects transcriptionfrom an inducible transcriptional regulatory element. In response toexposure to an inducing agent, transcription from the element generallyis initiated de novo or is increased above a basal or constitutive levelof expression. An inducing agent can be, for example, a stress conditionto which a cell is exposed, for example, a heat or cold shock, a toxicagent such as a heavy metal ion, or a lack of a nutrient, hormone,growth factor, or the like; or can be exposure to a molecule thataffects the growth or differentiation state of a cell such as a hormoneor a growth factor.

[0053] A nucleotide sequence operatively linked to a recombinantpolynucleotide of the invention also can encode an amino acid sequencethat is heterologous to the Her2/neu sequence. The heterologous aminoacid sequence can be a peptide, for example, a cell compartmentalizationdomain, which can facilitate localization of the Her2/neu target antigento a specific compartment of a cell, for example, to the cytosol,nucleus, plasma membrane, endoplasmic reticulum, mitochondrial membraneor matrix, chloroplast membrane or lumen, medial trans-Golgi cisternae,or a lysosome or endosome; a membrane translocating peptide, which canfacilitate transport of a Her2/neu target antigen across a cell membraneand into an intact cell; or a secretory peptide, which can facilitatesecretion of a Her2/neu target antigen out of a cell (see, for example,Hancock et al., EMBO J. 10:4033-4039, 1991; Buss et al., Mol. Cell.Biol. 8:3960-3963, 1988; U.S. Pat. No. 5,776,689, each of which isincorporated herein by reference).

[0054] A heterologous amino acid sequence also can be a peptide thatacts as a tag for detecting the presence of a fusion protein containingthe Her2/neu target antigen. Peptide tags, which are well known and canbe conveniently synthesized or expressed from an encodingpolynucleotide, include, for example, a His-6 tag, which can be detectedusing a divalent cation such as nickel ion or cobalt ion; a FLAGepitope, which can be detected using an anti-FLAG antibody (see, forexample, Hopp et al., BioTechnology 6:1204 (1988); U.S. Pat. No.5,011,912, each of which is incorporated herein by reference); a c-mycepitope, which can be detected using an antibody specific for theepitope; biotin, which can be detected using streptavidin or avidin; andglutathione S-transferase, which can be detected using glutathione. Suchtags can provide the additional advantage that they can facilitateisolation of the operatively linked Her2/neu target antigen.

[0055] A heterologous amino acid sequence operatively linked to aHer2/neu target antigen also can be an immunostimulatory polypeptide,which can further stimulate an immune response induced by the Her2/neutarget antigen. Immunostimulatory polypeptides include, for example, theB7 co-stimulatory molecules, and cytokines such as an interleukin, aninterferon, or GM-CSF. The heterologous amino acid sequence also can beany polypeptide that can contribute to the health or well being of asubject or that constitutes part of a treatment protocol of the subject,including, for example, a polypeptide hormone or hormone analog, achemokine or other cell or tissue growth factor.

[0056] The present invention also relates to a vector, which contains arecombinant polynucleotide of the invention, which encodes a Her2/neutarget antigen. The vector can be a cloning vector, which can be usefulwhere it is desired to manipulate the recombinant polynucleotide, forexample, by operatively linking a nucleotide sequence encoding apolypeptide of interest, or can be an expression vector, which generallycontains a promoter sequence and can contain a poly-A recognitionsequence, a ribosome recognition site or internal ribosome entry site,or other regulatory element such as an enhancer. In addition, a vectorgenerally contains elements required for replication in a prokaryotic oreukaryotic host system or both. Such vectors, which include plasmidvectors and viral vectors such as bacteriophage, baculovirus,retrovirus, lentivirus, adenovirus, vaccinia virus, semliki forest virusand adeno-associated virus vectors, are well known and can be purchasedfrom a commercial source (Promega, Madison Wis.; Stratagene, La JollaCalif.; GIBCO/BRL, Gaithersburg Md.) or can be constructed by oneskilled in the art (see, for example, Meth. Enzymol. Vol. 185, Goeddel,ed. (Academic Press, Inc., 1990); Jolly, Canc. Gene Ther. 1:51-64, 1994;Flotte, J. Bioenerg. Biomemb. 25:37-42, 1993; Kirshenbaum et al., J.Clin. Invest. 92:381-387, 1993; each of which is incorporated herein byreference).

[0057] A vector useful for containing a recombinant polynucleotide ofthe invention can be a plasmid vector or a viral vector. Methods ofmaking a viral vector containing a recombinant polynucleotide are knownin the art (see Larrick and Burck, Gene Therapy, Applications ofMolecular Biology (Elsevier, N.Y., 1991), which is incorporated hereinby reference). If desired, the viral vector can be replicationdefective, in which case an appropriate helper cell line is required toproduce viral particles containing the viral vector. A viral vector canbe based, for example, on an alphavirus, a retrovirus, an adenovirus, anadeno-associated virus, a vaccinia virus, or the like.

[0058] A retrovirus vector, for example, can be made by a retrovirusproducer cell, which, in turn, can be made by transfecting a plasmid,such as pLXSN into a retrovirus packaging cell line such as PA317 (see,Miller and Rosman, BioTechniques 7:980-990, 1989; Miller and Buttimore,Mol. Cell. Biol. 6:2895-2902, 1986, each of which is incorporated hereinby reference). A cell transfected with pLXSN is resistant to neomycinand, therefore, a stably transfected colony can be selected in a culturemedia supplemented with active neomycin, for example G418. Neomycinresistant cell lines can be identified by screening them for theproduction of replication defective murine leukemia retroviruses usingknown methods (see, Cepko, In Current Protocols in Molecular Biology,Suppl. 17, pp. 9.11.5-9.11.12, (Wiley-Interscience, NY 1992), which isincorporated herein by reference).

[0059] A retrovirus vector can be made by inserting a recombinantpolynucleotide of the invention into the selected vector plasmid. Therecombinant polynucleotide can be operatively linked to expressioncontrol sequences prior to introduction into the vector plasmid, or thevector can contain the required regulatory elements, to which therecombinant polynucleotide is operatively linked upon insertion into thevector plasmid. The retrovirus vector then is transfected into anappropriate helper cell line such that vectors containing therecombinant polynucleotide are produced. Additional methods of makingretrovirus vectors are known in the art (see, U.S. Pat. No. 5,399,347;U.S. Pat. No. 5,532,220; U.S. Pat. No. 5,240,846; Ram et al., CancerRes. 53:83-88, 1993, each of which is incorporated herein by reference).

[0060] In addition to retroviral vectors, other viral vectors can bemade and used for purposes of the present invention. For example,methods of making recombinant adenoviral vectors are well known (see,Karlsson et al., EMBO J. 5:2377-2385, 1986; Kleinerman et al., CancerRes. 55:2831-2836, 1995; Hamada et al., Gynecol. Oncol., 63:219-227,1996; Nabel et al., Science, 249:1285-1288, 1990; Berkner, BioTechniques6:616-629, 1989, each of which is incorporated herein by reference).Other recombinant viral vectors, such as adeno-associated virus vector,herpesvirus vectors, and vaccinia virus vectors are well known in theart (see Culver, Gene Therapy, A Handbook for Physicians (MaryAnnLiebert, Inc., NY 1994), which is incorporated herein by reference; see,also, Larrick and Burck, supra, 1991).

[0061] An alphavirus vector such as a Venezuelan equine encephalitisvirus (VEE) vector (AlphaVax, Inc.; Durham N.C.; see U.S. Pat. Nos.5,792,462, and 6,008,035, each of which is incorporated herein byreference) can be particularly useful where the recombinantpolynucleotide of the invention is to be used for administration to asubject in order to stimulate an immune response. Alphavirus VEE vectorshave been used to successfully vaccinate rodents and non-human primatesagainst influenza, Lassa fever, Ebola virus, Marburg virus, andbotulinum toxin (U.S. Pat. No. 5,643,576; Hevey et al., Virology251:28-37, 1998, each of which is incorporated herein by reference; see,also, Pushko et al., supra, 1997). VEE vectors have particularadvantages over other vaccine technologies, including they direct highantigen expression levels and show sustained efficacy over multiplesimultaneous or sequential innoculations of the vector. In addition, VEEvectors are not likely to be subject to an existing vector-specificimmune response, particularly in a human patient.

[0062] The present invention also provides a cell containing arecombinant polynucleotide or a vector of the invention. Such a cell canbe a prokaryotic cell or eukaryotic cell, particularly a mammalian cell.Where the cell contains a vector of the invention, the cell can be hostcell that is useful, for example, for propagating the vector, includingthe recombinant polynucleotide of the invention, or for expressing anencoded Her2/neu target antigen. Where the vector is a viral vectorplasmid, the cell also can be a helper cell, which provides in trans anyfactors necessary for replication and packaging of viral particlescontaining the viral vector. A cell containing a recombinantpolynucleotide of the invention also can be an immunoeffector cell, forexample, an APC such as a dendritic cell, in which the encoded Her2/neutarget antigen can be expressed and processed for presentation to Tcells, or can be a cell from which the encoded Her2/neu target antigenis secreted, such a cell being useful for stimulating an immunoeffectorcell such as an APC by providing the APC with the target antigen.

[0063] The present invention also relates to a recombinant polypeptide,which includes a Her2/neu target antigen having an amino acid sequencecorresponding to about amino acid residues 634 to 683 operatively linkedto amino acid residues about 1035 to 1255 of SEQ ID NO:1, or to aboutamino acid residues 635 to 685 operatively linked to about amino acidresidues 1037 to 1257 of SEQ ID NO:2, or which includes a Her2/neutarget antigen consisting of an amino acid sequence corresponding toamino acid residues 606 to 683 operatively linked to amino acid residues1035 to 1255 of SEQ ID NO:1, or to amino acid residues 608 to 685operatively linked to amino acid residues 1037 to 1257 of SEQ ID NO:2. Apolypeptide of the invention is exemplified by a Her2/neu target antigenhaving an amino acid sequence as set forth in SEQ ID NO:4, or an aminoacid sequence corresponding thereto, for example, as set forth in SEQ IDNO:10. A recombinant polypeptide of the invention can be expressed froma recombinant polynucleotide of the invention or can be chemicallysynthesized. As such, the Her2/neu target antigen can contain, forexample, one or more D-amino acids in place of a corresponding L-aminoacid; or can contain one or more amino acid analogs, for example, anamino acid that has been derivatized or otherwise modified at itsreactive side chain. Similarly, one or more peptide bonds in theHer2/neu target antigen can be modified, or a reactive group at theamino terminus or the carboxy terminus or both can be modified. Suchmodified Her2/neu target antigens can have improved stability to aprotease, an oxidizing agent or other reactive material the polypeptidemay encounter in a biological environment such as a tissue culturemedium or in a living subject and, therefore, can be particularly usefulin performing a method of the invention.

[0064] The present invention also provides a method for expressing aHer2/neu target antigen in a cell. Such a method can be performed, forexample, by contacting the cell with the recombinant polynucleotide, forexample, a nucleic acid molecule comprising SEQ D NO: 4 or SEQ ID NO:9or encoding SEQ ID NO:4 or SEQ ID NO:10, under conditions that allowexpression of the encoded Her2/neu target antigen by the cell. Thecontacting is performed under conditions such that the recombinantpolynucleotide is introduced into the cell, wherein that the encodedHer2/neu target antigen can be expressed.

[0065] Methods for introducing a polynucleotide into a cell are wellknown in the art and include, for example, transfection, lipofection,microinjection, electroporation, ballistic methods, and, with viralvectors, infection; and can include the use of liposomes, microemulsionsor the like, which can facilitate introduction of the polynucleotideinto the cell and can protect the polynucleotide from degradation priorto its introduction into the cell (see, for example, Sambrook et al.,Molecular Cloning: A laboratory manual (Cold Spring Harbor LaboratoryPress 1989); Ausubel et al., Current Protocols in Molecular Biology(John Wiley & Sons, Baltimore Md. 1987, and supplements), each of whichis incorporated herein by reference). For example, the recombinantpolynucleotide, which can be in a vector, can be incorporated into aliposome, which can be made target cell specific, if desired, byincorporating, for example, a lipid-conjugated antibody or otherreceptor or ligand specific for a particular target cell (see, forexample, Nabel et al., Proc. Natl. Acad. Sci. USA, 90:11307-11311(1993); Holmberg et al., J. Liposome Res., 1:393-406 (1990), each whichis incorporated herein by reference). The selection of a particularmethod for introducing the recombinant polynucleotide into a cell willdepend, for example, on the cell into which the polynucleotide is to beintroduced, as well as whether the cell is isolated in culture, or is insitu in a subject.

[0066] Introduction of a polynucleotide into a cell by infection with aviral vector is particularly advantageous in that it can efficientlyintroduce the nucleic acid molecule into a cell ex vivo or in vivo (see,for example, U.S. Pat. No. 5,399,346, which is incorporated herein byreference). Moreover, viruses are very specialized and can be selectedas vectors based on an ability to infect one or a few specific celltypes. Thus, their natural specificity can be used to target therecombinant polynucleotide to specific cell types. As such, a vectorbased on an HIV can be used to infect T cells, a vector based on anadenovirus can be used, for example, to infect respiratory epithelialcells, a vector based on a herpesvirus can be used to infect neuronalcells, and the like. Other vectors, such as adeno-associated viruses canhave greater host cell range and, therefore, can be used to infectvarious cell types, although viral or non-viral vectors also can bemodified with specific receptors or ligands to alter target specificitythrough receptor mediated events. As described above, alphavirus VEEvectors have been used to successfully vaccinate rodents and non-humanprimates against various infective viruses and toxins and, therefore,can be particularly useful for practicing the methods of the invention.

[0067] The cell to be contacted can be any cell in which the Her2/neutarget antigen can be expressed, including prokaryotic and eukaryoticcells, and particularly mammalian cells. The cell can be contacted withthe recombinant polynucleotide in culture, in which case the cell can beone that that has been established for passage in tissue culture. Wherethe cells are adapted to tissue culture, the cells expressing a Her2/neutarget antigen can comprise a panel of such cells, for example, a panelof dendritic cells having different but known haplotypes. Such panels ofcells can be useful as a convenient source from which a clinician canselect a particular dendritic cells for administration to a patient inwhom it is desired to stimulate an immune response against cellsexpressing Her2/neu. In addition, the cell can be one or a population ofcells that has been removed from the subject and is contacted ex vivo,or the contacting can be performed in vivo. Where the contacting isperformed in vivo, the recombinant polynucleotide can be administered tothe site of cell or to a site to which the cell can migrate, or can beadministered systemically such that it can circulate to the cells inwhich the Her2/neu target antigen is expressed. In addition, therecombinant polynucleotide can, but need not, be contained in a vector,which can be a viral vector. In general, the recombinant polynucleotideis operatively linked to expression regulatory elements required fortranscription and translation, although such elements can be a componentof a vector containing the recombinant polynucleotide.

[0068] The present invention also provides a method of isolating aHer2/neu target antigen from a cell expressing the target antigen. Sucha method can be performed by contacting the cell with a recombinantpolynucleotide of the invention under conditions that allow expressionof the encoded Her2/neu target antigen by the cell, and isolating theexpressed Her2/neu target antigen from the cell. The Her2/neu targetantigen can be isolated from the cell using any convenient method,including, for example, by an affinity chromatography method such asimmunoaffinity chromatography, a gel filtration chromatography method orby gel electrophoresis of a protein extract prepared from cellsexpressing the Her2/neu target antigen (see, for example, Deutscher,Guide to Protein Purification, Academic Press, Inc., 1990, which isincorporated herein by reference). An affinity chromatography method,for example, can be based on the binding of a peptide operatively linkedto the Her2/neu target antigen, for example, a polyhistidine tagpeptide, which can be bound by a nickel ion chelate. If desired, apolypeptide such as a peptide tag that is operatively linked to aHer2/neu target antigen of the invention can further contain a cleavagesite positioned between the target antigen and the peptide, for example,a protease or chemical cleavage site, which is not also present in theHer2/neu target antigen, such that, upon isolation of the fusionprotein, the Her2/neu target antigen can be released from theoperatively linked peptide or polypeptide. Accordingly, the presentinvention also provides an isolated Her2/neu target antigen obtained bysuch a method.

[0069] The present invention provides a method of obtaining a cell thatis genetically modified to express a Her2/target antigen by contacting acell with a recombinant polynucleotide of the invention under conditionsthat allow expression of the encoded Her2/neu target antigen by thecell, and isolating a cell expressing the Her2/neu target antigen. Suchcells can be isolated, for example, by including the recombinantpolypeptide in a vector containing a selectable marker such as anantibiotic resistance gene, or by operatively linking a nucleotidesequence encoding a selectable marker to the recombinant polypeptide,such that cells expressing the marker and, therefore, the Her2/neutarget antigen, can be selected. Cells selected by such a method canfurther be cloned, for example, by performing a limiting dilution of thecells. Accordingly, the present invention further provides an isolatedcell, which expresses the Her2/neu target antigen, obtained by such amethod.

[0070] The present invention also provides methods of stimulating animmune response in a subject against cells that express Her2/neu. Thecells that express Her2/neu can be cancer cells or any other cells thatexpress Her2/neu, particularly cells involved in a pathologic condition.The disclosed methods are particularly useful for stimulating an immuneresponse against cells that are involved in a pathologic condition andoverexpress Her2/neu as compared to corresponding cells that are notinvolved in the pathologic condition. For example, the cells can becancer cells that overexpress Her2/neu as compared to the level ofHer2/neu expressed by normal cell counterparts to the cancer cells. Inone embodiment, a method of stimulating an immune response in a subjectagainst cancer cells that express Her2/neu is performed by contactingcells with a recombinant polynucleotide of the invention underconditions that allow expression from the recombinant polynucleotide ofthe Her2/neu target antigen in the cells. The cells that are contactedwith the recombinant polypeptide can be autologous or allogeneic withrespect to the subject to be treated, and, where autologous, can becontacted ex vivo or in vivo. In addition, the cells that are contactedwith the recombinant polynucleotide can be antigen presenting cells(APCs) or can be cells that can express and secrete the Her2/neu targetantigen such that it can be taken up and processed by an APC.

[0071] A cell to be contacted with a recombinant polynucleotide of theinvention can be contacted in a culture medium, generally a tissueculture medium or other physiologically acceptable medium that isconducive to survival of the cells at least for the time required tointroduce the recombinant polynucleotide into the cell, for example, aphysiological saline solution. Where the cell that is contacted with therecombinant polynucleotide is an APC, a method of stimulating an immuneresponse is performed by administering the APCs, which are expressingthe Her2/neu target antigen, to the subject, such that the APCs canpresent the processed antigen to immunoeffector cells. Where the cellthat is contacted with the recombinant polynucleotide is a cell otherthan an APC, for example, a fibroblast, the recombinant polynucleotidecan further include an operatively linked nucleotide sequence encoding asecretory peptide, which allows secretion of the Her2/neu target antigenfrom the cell. The cell expressing (and secreting) the Her2/neu targetantigen then is contacted with an APC, either in culture, or byadministering the cell to the subject to be treated such that an APC inthe subject can take up and process the target antigen for presentationto other immunoeffector cells, including Th cells and CTLs. Where thecells that express the Her2/neu target antigen and the APCs arecontacted in culture, APCs that have endocytosed the Her2/neu targetantigen, and, if desired, the cells expressing the Her2/neu targetantigen are administered to the subject to be treated.

[0072] A cell to be contacted with a recombinant polynucleotide of theinvention also can be contacted in vivo. The cells to be contacted invivo can be APCs, cells other than APCs, which can secrete the Her2/neutarget antigen such that APCs can endocytose and process the targetantigen, or a combination of such cells. The recombinant polynucleotide,which generally is formulated as a pharmaceutical composition, can beadministered to the subject as a naked DNA molecule, which can becontained in a vector, or can be formulated in a liposome or othermatrix that facilitates uptake of the recombinant polynucleotide by acell, or can be contained in a viral particle, for example, a VEEreplicon particle (AlphaVax, Inc.).

[0073] In another embodiment, a method of immunostimulatingimmunoeffector cells against cancer cells expressing Her2/neu isperformed by contacting the immunoeffector cells, particularly APCs,with a recombinant Her2/neu target antigen polypeptide. Theimmunoeffector cells can be any cells that can be immunostimulated dueto contact with the recombinant polypeptide, particularly antigenpresenting cells such as dendritic cells, B cells, and the like. Thecontacting can be performed on cells in culture, which can be autologouscells or allogeneic cells, or can be performed in vivo by administeringthe recombinant polypeptide to the subject. Where the cells arecontacted in culture, the immunostimulated cells can be isolated, ifdesired. As such, the present invention also provides a method ofisolating immunoeffector cells that have been immunostimulated due tocontact with a Her2/neu target antigen, and further provides an isolatedimmunostimulated cell isolated by such a method.

[0074] For administration to a subject, a recombinant Her2/neu targetantigen or an encoding recombinant polynucleotide generally isformulated as a pharmaceutical composition. Accordingly, the presentinvention provides a pharmaceutical composition, which generallycontains, in addition to the recombinant polynucleotide or polypeptideof the invention, a pharmaceutically acceptable carrier, for example, anaqueous solution such as physiologically buffered saline or othersolvent or vehicle such as a glycol, glycerol, an oil such as olive oilor an injectable organic esters. A pharmaceutically acceptable carrieralso can include a physiologically acceptable compound that acts, forexample, to stabilize the Her2/neu target antigen or encodingrecombinant polynucleotide or to increase its absorption.Physiologically acceptable compounds include, for example,carbohydrates, such as glucose, sucrose or dextrans, antioxidants, suchas ascorbic acid or glutathione, chelating agents, low molecular weightproteins or other stabilizers or excipients. Similarly, a cell that hasbeen treated in culture for purposes of the practicing the methods ofthe invention also can be formulated in a pharmaceutical compositionwhen the cells are to be administered to a subject.

[0075] One skilled in the art would know that the choice of apharmaceutically acceptable carrier, including a physiologicallyacceptable compound, depends, for example, on whether a Her2/neu targetantigen polypeptide or an encoding polynucleotide is to be administered,as well as on the route of administration of the composition. Where thepharmaceutical composition is administered as a vaccine, it generally isadministered intramuscularly, intradermally, or subcutaneously. However,it also can be administered orally or parenterally such asintravenously, and can be administered by injection, intubation, orother such method known in the art.

[0076] A pharmaceutical composition of the invention also can contain asecond reagent such as a diagnostic reagent, nutritional substance,toxin, or therapeutic agent, for example, a cancer chemotherapeuticagent, or an immunostimulatory agents such as a cytokine or a B7molecule In addition, a pharmaceutical composition containing arecombinant polypeptide Her2/neu target antigen can contain an adjuvant,for example, alum, DETOX adjuvant (Ribi Immunochem Research, Inc.;Hamilton Mont.), or Freund's complete or incomplete adjuvant. Theaddition of an adjuvant can enhance the immunogenicity of a Her2/neutarget antigen, thus decreasing the amount of target antigen required tostimulate an immune response. Adjuvants can augment the immune responseby prolonging antigen persistence, enhancing co-stimulatory signals,inducing granuloma formation, stimulating lymphocyte proliferationnonspecifically, or improving apposition of a T cell and an APC.

[0077] A recombinant polynucleotide or polypeptide of the invention alsocan be incorporated within an encapsulating material such as into anoil-in-water emulsion, a microemulsion, micelle, mixed micelle,liposome, microsphere or other polymer matrix (see, for example,Gregoriadis, Liposome Technology, Vol. 1 (CRC Press, Boca Raton, Fla.1984); Fraley, et al., Trends Biochem. Sci., 6:77, 1981, each of whichis incorporated herein by reference). Liposomes, for example, whichconsist of phospholipids or other lipids, are nontoxic, physiologicallyacceptable and metabolizable carriers that are relatively simple to makeand administer. “Stealth” liposomes (see, for example, U.S. Pat. Nos.5,882,679; 5,395,619; and 5,225,212, each of which is incorporatedherein by reference) are an example of such encapsulating material.Cationic liposomes, for example, also can be modified with specificreceptors or ligands (Morishita et al., J. Clin. Invest., 91:2580-2585,1993, which is incorporated herein by reference). In addition, apolynucleotide agent can be introduced into a cell using, for example,adenovirus-polylysine DNA complexes (see, for example, Michael et al.,J. Biol. Chem. 268:6866-6869, 1993, which is incorporated herein byreference).

[0078] The total amount of a pharmaceutical composition to beadministered in practicing a method of the invention can be administeredto a subject as a single dose, either as a bolus or by infusion over arelatively short period of time, and can be followed up with one or morebooster doses over a period of time. One skilled in the art would knowthat the amount of the pharmaceutical composition to stimulate an immuneresponse in a subject depends on various factors including the age andgeneral health of the subject, as well as the route of administrationand the number of treatments to be administered. In view of thesefactors, the skilled artisan would adjust the particular dosage asnecessary. In general, the formulation of the pharmaceutical compositionand the routes and frequency of administration are determined,initially, using Phase I and Phase II clinical trials.

[0079] The present invention also relates to a method of making a targetantigen, which can stimulate an immune response against a proteinexpressed on the surface of a cancer cell. Such a method can beperformed, for example, by selecting a first peptide portion of theprotein, wherein the first peptide portion includes the transmembranedomain and has limited homology to proteins other than the proteinexpressed on the surface of the tumor cell; selecting a second peptideportion of the protein, which includes a portion of the cytoplasmicdomain of the protein and has limited homology to proteins other thanthe protein expressed on the surface of the tumor cell; and operativelylinking the first peptide portion and the second peptide portion,thereby making the target antigen. As used herein, the term “limitedhomology” is used relatively to indicate that a reference peptide havinga defined amino acid sequence has less than about 30% sequence identitywith proteins to which it is being compared. In general, the lesshomology a sequence of a reference peptide (i.e., a peptide portion of aprotein to be used for constructing a target antigen) has with proteinsthat are normally expressed in a subject to which the target antigen isto be administered, the better suited the peptide is for making thetarget antigen. Such limited homology can be determined using readilyavailable search algorithms, such as BLASTP and the like.

[0080] The following example is intended to illustrate but not limit theinvention.

EXAMPLE 1 Her2/neu Vaccine Protects Against Tumor Growth

[0081] This example demonstrates that a vaccine based on regions of theHer2/neu protein that are not present in other proteins increasesurvival in a syngeneic rat tumor model.

[0082] The Her 2/neu breast cancer tumor associated antigen (TAA) wasselected as the prototypical target antigen for the development andevaluation of genetic vaccination strategies. The Her2/neu protein is amember of the c-erb B/epidermal growth factor receptor family and isoverexpressed in approximately 35% of breast cancers (Harris et. al.,Cancer of the Breast, in “CANCER: Principles & Practice of Oncology”(Devita and Rosenberg, Eds., J.B. Lippincott Co., Philadelphia Pa.,1993)). The Her2/neu protein was selected as a target antigen becauseimmune recognition of this TAA has been reported (Disis et al., Clin.Cancer Res. 5:1289-1297, 1999; Tuttle et al., Clin. Cancer Res.4:2015-2024, 1998, each of which is incorporated herein by reference),and because, if a robust anti-TAA immune response was generated using agenetic vaccination strategy, the critical issues of specificity ofresponse and potential cross-reactive auto-immune phenomena could beaddressed.

[0083] Two regions of the human Her 2/neu molecule demonstrating thelowest degree of homology with other known normal human proteins wereselected as the putative target antigen sequence for a geneticvaccination strategy. The selected regions include a 49 amino acidsequence encompassing the transmembrane domain (amino acids 634 to 683of SEQ ID NO:1) and a 221 amino acid sequence of the intracellulardomain (amino acids 1035 to 1255 of SEQ ID NO:1). When combined to forma single polypeptide, the new sequence did not have any significanthomology with normal proteins when examined using a BLAST search throughNCBI and a FASTA search covering SwissPro and other databases. Thistarget sequence, which contain some of the previously identified T cellepitopes (Disis et al., supra, 1999), was selected to minimize anypotential cross-reactive, autoimmune responses and to maximize thespecificity of the elicited immune response. Because this sequence ispredominantly intracellular and represents a truncated or partialprotein sequence, it should be rapidly degraded and presented in thecontext of MHC class 1 and, therefore, available for CTL recognition(Rock and Goldberg, Ann. Rev. Immunol. 17:739-779, 1999, which isincorporated herein by reference).

[0084] The high degree of homology between human Her 2/neu and rat neusequences (Yamamoto et al., Nature 319:230-4, 1986, which isincorporated herein by reference) allowed for the construction of anentirely homologous sequence of rat neu for use as a putative targetantigen sequence in an established rat model of mammary carcinoma. Therat mammary tumor cell line, 13762 MAT BIII (ATCC, CRL 1666), wasderived from the Fisher 344 strain and represents a syngeneic tumor line(Segaloff, Recent Prog. Horm. Res. 22:351-379, 1966, which isincorporated herein by reference). Subcutaneous (SC) inoculation of5×10⁴ tumor cells results in the establishment of a tumor nodule inapproximately 10 days with sufficient growth requiring euthanization ofuntreated animals in 5-8 days. This tumor expresses low levels of ratMHC class 1 and modest levels of rat neu as evaluated by flow cytometry.The low level expression of MHC class 1 in the tumor model recapitulatesthe findings in human breast cancer (Pistillo et al., Hum. Immunol.61:397-407, 2000; Vitale et al., Cancer Res. 58:737-42, 1998, each ofwhich is incorporated herein by reference). In view of this result,coupled with the modest overexpression of rat neu and the aggressivegrowth characteristics of this tumor, this model system provides arigorous, entirely syngeneic model of breast cancer for testingimmunotherapeutic strategies.

[0085] It is noted that rat neu transgenic mouse models exist, includingmodels that use an activated and transforming mutated rat neu (Bouchardet al., Cell 57:931-936, 1989; Guy et al., J. Biol. Chem. 271:7673-7678,1996; Muller et al., Cell 54:105-115, 1988, each of which isincorporated herein by reference) or that use a putative wild type ratneu, which may have undergone activating mutations, under the control ofvarious promoters. As with most transgenic models, however, it isunclear whether expression of the transgene is regulated in a manneridentical to that seen in the natural host. The Fisher 344 rat model wasselected due, in part, to these concerns.

[0086] The target antigen sequence was derived from 13762 tumor mRNAusing reverse transcription-PCR. The nucleotide sequence encoding thetransmembrane fragment of the target antigen is shown in FIG. 6A(proximal rat Her2/neu polypeptide; SEQ ID NO:6). The amplified sequencecontains an introduced BspE1 site (underlined), which was encoded by thePCR primers. FIG. 6B shows the nucleotide sequence encoding the terminalcytoplasmic fragment (SEQ ID NO:8), again with the introduced BspE1 siteunderlined. FIG. 6A also shows the recombinant polynucleotide (SEQ IDNO:3) and encoded Her2/neu target antigen (SEQ ID NO: 5), with theadditional sequences added to initiate translation and maintain readingframe double underlined and the fusion site, including an additionalinserted serine residue underlined. SEQ ID NO:3 was prepared by PCRamplification of a template prepared by linking SEQ ID NOS:6 and 8 atthe BspE1 site (underlined). The coding sequence of the Her2/neu antigen(SEQ ID NO:3) and the amino acid sequence (SEQ ID NO:4) of the encodedantigen are shown (FIG. 1). FIG. 2 shows the corresponding nucleotideand amino acid sequences of human Her2/neu (SEQ ID NOS:9-14).

[0087] The nucleotide sequence encoding the Her2/neu target antigen (SEQID NO:3) was cloned into the polynucleotide vaccine vector, pITL(Internatl. Publ. No. WO 98/06863). Purified plasmid (with or withoutthe target antigen sequence) containing endotoxin levels less than 5 EUper mg DNA was produced (Prieto et al., BioTechniques 29:1204-1206,2000, which is incorporated herein by reference). Animals received threeseparate intramuscular vaccinations of 100 μg of covalently closedcircular plasmid DNA at three week intervals. Positive control animalsreceived 1×10⁶ irradiated (20 cGy) 13762 tumor cells in the subcutaneousspace on the same schedule. A tumor challenge of 5×10⁴ viable tumorcells was administered subcutaneously 3 weeks after completion of thevaccination regimen. Animals developing tumors were euthanized at thedesignated maximal tumor dimension.

[0088] Administration of the target antigen DNA vaccine resulted in astatistically significant (p=0.02; log rank analysis) delay in tumoroutgrowth (FIG. 2), but no protection was observed; all animalsdeveloped tumors. The DNA vaccine regimen also resulted in a significanttumor-specific proliferative response.

[0089] The target antigen coding sequence (SEQ ID NO:5) also was clonedinto the construct for inclusion in the Venezuelan equine encephalitisvirus (VEE) replicon particles (VRP; see U.S. Pat. Nos. 5,792,462, and6,008,035); this vector was designated VRP-rNeu. VRP containing theinfluenza hemagglutinin antigen (VRP-HA) was used as a specificitycontrol. Very similar results were obtained in two separate experiments.Intramuscular or subcutaneous injection of 1×10⁷ infectious units (ifu)of VRP-rNeu, administered every three weeks for a total of threeinjections, resulted in approximately 50% survival without tumor aftertumor challenge in excess of 120 days, (FIG. 3; see, also FIG. 4A,showing growth of tumors in individual animals receiving 1×10⁷ ifu,intramuscularly). Lower doses (1×10⁶ ifu or 1×10⁵ ifu) of VRPs also wereadministered subcutaneously. As shown in FIGS. 4B and 4C, tumor growthon average was delayed in an apparently dose-dependent relationship.

[0090] Tumor-free survival was a result of both overt protection, seenonly in the IM route, and in permanent regression of tumors in 25-50% ofthe animals. This regression suggested that there was an immunologicbasis for the survival benefit. Accordingly, surviving animals werere-challenged 120 days after the initial tumor challenge with anidentical dose of viable 13762 tumor cells. All vaccinated animalsrejected the tumor challenge, while control animals developed tumors(FIG. 5), indicating that these animals have immunologic memory.

[0091] These results indicate that a genetic vaccine encoding theHer2/neu target shown as SEQ ID NO:4 reduced or inhibited breast cancercell growth in a syngeneic animal model, and that the effectiveness ofthe vaccination is due to an immunologic response in the animals.

[0092] Although the invention has been described with reference to theabove example, it will be understood that modifications and variationsare encompassed within the spirit and scope of the invention.Accordingly, the invention is limited only by the following claims.

1 14 1 1255 PRT Homo sapiens 1 Met Glu Leu Ala Ala Leu Cys Arg Trp GlyLeu Leu Leu Ala Leu Leu 1 5 10 15 Pro Pro Gly Ala Ala Ser Thr Gln ValCys Thr Gly Thr Asp Met Lys 20 25 30 Leu Arg Leu Pro Ala Ser Pro Glu ThrHis Leu Asp Met Leu Arg His 35 40 45 Leu Tyr Gln Gly Cys Gln Val Val GlnGly Asn Leu Glu Leu Thr Tyr 50 55 60 Leu Pro Thr Asn Ala Ser Leu Ser PheLeu Gln Asp Ile Gln Glu Val 65 70 75 80 Gln Gly Tyr Val Leu Ile Ala HisAsn Gln Val Arg Gln Val Pro Leu 85 90 95 Gln Arg Leu Arg Ile Val Arg GlyThr Gln Leu Phe Glu Asp Asn Tyr 100 105 110 Ala Leu Ala Val Leu Asp AsnGly Asp Pro Leu Asn Asn Thr Thr Pro 115 120 125 Val Thr Gly Ala Ser ProGly Gly Leu Arg Glu Leu Gln Leu Arg Ser 130 135 140 Leu Thr Glu Ile LeuLys Gly Gly Val Leu Ile Gln Arg Asn Pro Gln 145 150 155 160 Leu Cys TyrGln Asp Thr Ile Leu Trp Lys Asp Ile Phe His Lys Asn 165 170 175 Asn GlnLeu Ala Leu Thr Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys 180 185 190 HisPro Cys Ser Pro Met Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser 195 200 205Ser Glu Asp Cys Gln Ser Leu Thr Arg Thr Val Cys Ala Gly Gly Cys 210 215220 Ala Arg Cys Lys Gly Pro Leu Pro Thr Asp Cys Cys His Glu Gln Cys 225230 235 240 Ala Ala Gly Cys Thr Gly Pro Lys His Ser Asp Cys Leu Ala CysLeu 245 250 255 His Phe Asn His Ser Gly Ile Cys Glu Leu His Cys Pro AlaLeu Val 260 265 270 Thr Tyr Asn Thr Asp Thr Phe Glu Ser Met Pro Asn ProGlu Gly Arg 275 280 285 Tyr Thr Phe Gly Ala Ser Cys Val Thr Ala Cys ProTyr Asn Tyr Leu 290 295 300 Ser Thr Asp Val Gly Ser Cys Thr Leu Val CysPro Leu His Asn Gln 305 310 315 320 Glu Val Thr Ala Glu Asp Gly Thr GlnArg Cys Glu Lys Cys Ser Lys 325 330 335 Pro Cys Ala Arg Val Cys Tyr GlyLeu Gly Met Glu His Leu Arg Glu 340 345 350 Val Arg Ala Val Thr Ser AlaAsn Ile Gln Glu Phe Ala Gly Cys Lys 355 360 365 Lys Ile Phe Gly Ser LeuAla Phe Leu Pro Glu Ser Phe Asp Gly Asp 370 375 380 Pro Ala Ser Asn ThrAla Pro Leu Gln Pro Glu Gln Leu Gln Val Phe 385 390 395 400 Glu Thr LeuGlu Glu Ile Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro 405 410 415 Asp SerLeu Pro Asp Leu Ser Val Phe Gln Asn Leu Gln Val Ile Arg 420 425 430 GlyArg Ile Leu His Asn Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu 435 440 445Gly Ile Ser Trp Leu Gly Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly 450 455460 Leu Ala Leu Ile His His Asn Thr His Leu Cys Phe Val His Thr Val 465470 475 480 Pro Trp Asp Gln Leu Phe Arg Asn Pro His Gln Ala Leu Leu HisThr 485 490 495 Ala Asn Arg Pro Glu Asp Glu Cys Val Gly Glu Gly Leu AlaCys His 500 505 510 Gln Leu Cys Ala Arg Gly His Cys Trp Gly Pro Gly ProThr Gln Cys 515 520 525 Val Asn Cys Ser Gln Phe Leu Arg Gly Gln Glu CysVal Glu Glu Cys 530 535 540 Arg Val Leu Gln Gly Leu Pro Arg Glu Tyr ValAsn Ala Arg His Cys 545 550 555 560 Leu Pro Cys His Pro Glu Cys Gln ProGln Asn Gly Ser Val Thr Cys 565 570 575 Phe Gly Pro Glu Ala Asp Gln CysVal Ala Cys Ala His Tyr Lys Asp 580 585 590 Pro Pro Phe Cys Val Ala ArgCys Pro Ser Gly Val Lys Pro Asp Leu 595 600 605 Ser Tyr Met Pro Ile TrpLys Phe Pro Asp Glu Glu Gly Ala Cys Gln 610 615 620 Pro Cys Pro Ile AsnCys Thr His Ser Cys Val Asp Leu Asp Asp Lys 625 630 635 640 Gly Cys ProAla Glu Gln Arg Ala Ser Pro Leu Thr Ser Ile Ile Ser 645 650 655 Ala ValVal Gly Ile Leu Leu Val Val Val Leu Gly Val Val Phe Gly 660 665 670 IleLeu Ile Lys Arg Arg Gln Gln Lys Ile Arg Lys Tyr Thr Met Arg 675 680 685Arg Leu Leu Gln Glu Thr Glu Leu Val Glu Pro Leu Thr Pro Ser Gly 690 695700 Ala Met Pro Asn Gln Ala Gln Met Arg Ile Leu Lys Glu Thr Glu Leu 705710 715 720 Arg Lys Val Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val TyrLys 725 730 735 Gly Ile Trp Ile Pro Asp Gly Glu Asn Val Lys Ile Pro ValAla Ile 740 745 750 Lys Val Leu Arg Glu Asn Thr Ser Pro Lys Ala Asn LysGlu Ile Leu 755 760 765 Asp Glu Ala Tyr Val Met Ala Gly Val Gly Ser ProTyr Val Ser Arg 770 775 780 Leu Leu Gly Ile Cys Leu Thr Ser Thr Val GlnLeu Val Thr Gln Leu 785 790 795 800 Met Pro Tyr Gly Cys Leu Leu Asp HisVal Arg Glu Asn Arg Gly Arg 805 810 815 Leu Gly Ser Gln Asp Leu Leu AsnTrp Cys Met Gln Ile Ala Lys Gly 820 825 830 Met Ser Tyr Leu Glu Asp ValArg Leu Val His Arg Asp Leu Ala Ala 835 840 845 Arg Asn Val Leu Val LysSer Pro Asn His Val Lys Ile Thr Asp Phe 850 855 860 Gly Leu Ala Arg LeuLeu Asp Ile Asp Glu Thr Glu Tyr His Ala Asp 865 870 875 880 Gly Gly LysVal Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu Arg 885 890 895 Arg ArgPhe Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val 900 905 910 TrpGlu Leu Met Thr Phe Gly Ala Lys Pro Tyr Asp Gly Ile Pro Ala 915 920 925Arg Glu Ile Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro 930 935940 Pro Ile Cys Thr Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met 945950 955 960 Ile Asp Ser Glu Cys Arg Pro Arg Phe Arg Glu Leu Val Ser GluPhe 965 970 975 Ser Arg Met Ala Arg Asp Pro Gln Arg Phe Val Val Ile GlnAsn Glu 980 985 990 Asp Leu Gly Pro Ala Ser Pro Leu Asp Ser Thr Phe TyrArg Ser Leu 995 1000 1005 Leu Glu Asp Asp Asp Met Gly Asp Leu Val AspAla Glu Glu Tyr 1010 1015 1020 Leu Val Pro Gln Gln Gly Phe Phe Cys ProAsp Pro Ala Pro Gly 1025 1030 1035 Ala Gly Gly Met Val His His Arg HisArg Ser Ser Ser Thr Arg 1040 1045 1050 Ser Gly Gly Gly Asp Leu Thr LeuGly Leu Glu Pro Ser Glu Glu 1055 1060 1065 Glu Ala Pro Arg Ser Pro LeuAla Pro Ser Glu Gly Ala Gly Ser 1070 1075 1080 Asp Val Phe Asp Gly AspLeu Gly Met Gly Ala Ala Lys Gly Leu 1085 1090 1095 Gln Ser Leu Pro ThrHis Asp Pro Ser Pro Leu Gln Arg Tyr Ser 1100 1105 1110 Glu Asp Pro ThrVal Pro Leu Pro Ser Glu Thr Asp Gly Tyr Val 1115 1120 1125 Ala Pro LeuThr Cys Ser Pro Gln Pro Glu Tyr Val Asn Gln Pro 1130 1135 1140 Asp ValArg Pro Gln Pro Pro Ser Pro Arg Glu Gly Pro Leu Pro 1145 1150 1155 AlaAla Arg Pro Ala Gly Ala Thr Leu Glu Arg Pro Lys Thr Leu 1160 1165 1170Ser Pro Gly Lys Asn Gly Val Val Lys Asp Val Phe Ala Phe Gly 1175 11801185 Gly Ala Val Glu Asn Pro Glu Tyr Leu Thr Pro Gln Gly Gly Ala 11901195 1200 Ala Pro Gln Pro His Pro Pro Pro Ala Phe Ser Pro Ala Phe Asp1205 1210 1215 Asn Leu Tyr Tyr Trp Asp Gln Asp Pro Pro Glu Arg Gly AlaPro 1220 1225 1230 Pro Ser Thr Phe Lys Gly Thr Pro Thr Ala Glu Asn ProGlu Tyr 1235 1240 1245 Leu Gly Leu Asp Val Pro Val 1250 1255 2 1257 PRTRattus norvegicus 2 Met Glu Leu Ala Ala Trp Cys Arg Trp Gly Phe Leu LeuAla Leu Leu 1 5 10 15 Pro Pro Gly Ile Ala Gly Thr Gln Val Cys Thr GlyThr Asp Met Lys 20 25 30 Leu Arg Leu Pro Ala Ser Pro Glu Thr His Leu AspMet Leu Arg His 35 40 45 Leu Tyr Gln Gly Cys Gln Val Val Gln Gly Asn LeuGlu Leu Thr Tyr 50 55 60 Val Pro Ala Asn Ala Ser Leu Ser Phe Leu Gln AspIle Gln Glu Val 65 70 75 80 Gln Gly Tyr Met Leu Ile Ala His Asn Gln ValLys Arg Val Pro Leu 85 90 95 Gln Arg Leu Arg Ile Val Arg Gly Thr Gln LeuPhe Glu Asp Lys Tyr 100 105 110 Ala Leu Ala Val Leu Asp Asn Arg Asp ProGln Asp Asn Val Ala Ala 115 120 125 Ser Thr Pro Gly Arg Thr Pro Glu GlyLeu Arg Glu Leu Gln Leu Arg 130 135 140 Ser Leu Thr Glu Ile Leu Lys GlyGly Val Leu Ile Arg Gly Asn Pro 145 150 155 160 Gln Leu Cys Tyr Gln AspMet Val Leu Trp Lys Asp Val Phe Arg Lys 165 170 175 Asn Asn Gln Leu AlaPro Val Asp Ile Asp Thr Asn Arg Ser Arg Ala 180 185 190 Cys Pro Pro CysAla Pro Ala Cys Lys Asp Asn His Cys Trp Gly Glu 195 200 205 Ser Pro GluAsp Cys Gln Ile Leu Thr Gly Thr Ile Cys Thr Ser Gly 210 215 220 Cys AlaArg Cys Lys Gly Arg Leu Pro Thr Asp Cys Cys His Glu Gln 225 230 235 240Cys Ala Ala Gly Cys Thr Gly Pro Lys His Ser Asp Cys Leu Ala Cys 245 250255 Leu His Phe Asn His Ser Gly Ile Cys Glu Leu His Cys Pro Ala Leu 260265 270 Val Thr Tyr Asn Thr Asp Thr Phe Glu Ser Met His Asn Pro Glu Gly275 280 285 Arg Tyr Thr Phe Gly Ala Ser Cys Val Thr Thr Cys Pro Tyr AsnTyr 290 295 300 Leu Ser Thr Glu Val Gly Ser Cys Thr Leu Val Cys Pro ProAsn Asn 305 310 315 320 Gln Glu Val Thr Ala Glu Asp Gly Thr Gln Arg CysGlu Lys Cys Ser 325 330 335 Lys Pro Cys Ala Arg Val Cys Tyr Gly Leu GlyMet Glu His Leu Arg 340 345 350 Gly Ala Arg Ala Ile Thr Ser Asp Asn ValGln Glu Phe Asp Gly Cys 355 360 365 Lys Lys Ile Phe Gly Ser Leu Ala PheLeu Pro Glu Ser Phe Asp Gly 370 375 380 Asp Pro Ser Ser Gly Ile Ala ProLeu Arg Pro Glu Gln Leu Gln Val 385 390 395 400 Phe Glu Thr Leu Glu GluIle Thr Gly Tyr Leu Tyr Ile Ser Ala Trp 405 410 415 Pro Asp Ser Leu ArgAsp Leu Ser Val Phe Gln Asn Leu Arg Ile Ile 420 425 430 Arg Gly Arg IleLeu His Asp Gly Ala Tyr Ser Leu Thr Leu Gln Gly 435 440 445 Leu Gly IleHis Ser Leu Gly Leu Arg Ser Leu Arg Glu Leu Gly Ser 450 455 460 Gly LeuAla Leu Ile His Arg Asn Ala His Leu Cys Phe Val His Thr 465 470 475 480Val Pro Trp Asp Gln Leu Phe Arg Asn Pro His Gln Ala Leu Leu His 485 490495 Ser Gly Asn Arg Pro Glu Glu Asp Leu Cys Val Ser Ser Gly Leu Val 500505 510 Cys Asn Ser Leu Cys Ala His Gly His Cys Trp Gly Pro Gly Pro Thr515 520 525 Gln Cys Val Asn Cys Ser His Phe Leu Arg Gly Gln Glu Cys ValGlu 530 535 540 Glu Cys Arg Val Trp Lys Gly Leu Pro Arg Glu Tyr Val SerAsp Lys 545 550 555 560 Arg Cys Leu Pro Cys His Pro Glu Cys Gln Pro GlnAsn Ser Ser Glu 565 570 575 Thr Cys Phe Gly Ser Glu Ala Asp Gln Cys AlaAla Cys Ala His Tyr 580 585 590 Lys Asp Ser Ser Ser Cys Val Ala Arg CysPro Ser Gly Val Lys Pro 595 600 605 Asp Leu Ser Tyr Met Pro Ile Trp LysTyr Pro Asp Glu Glu Gly Ile 610 615 620 Cys Gln Pro Cys Pro Ile Asn CysThr His Ser Cys Val Asp Leu Asp 625 630 635 640 Glu Arg Gly Cys Pro AlaGlu Gln Arg Ala Ser Pro Val Thr Phe Ile 645 650 655 Ile Ala Thr Val ValGly Val Leu Leu Phe Leu Ile Leu Val Val Val 660 665 670 Val Gly Ile LeuIle Lys Arg Arg Arg Gln Lys Ile Arg Lys Tyr Thr 675 680 685 Met Arg ArgLeu Leu Gln Glu Thr Glu Leu Val Glu Pro Leu Thr Pro 690 695 700 Ser GlyAla Met Pro Asn Gln Ala Gln Met Arg Ile Leu Lys Glu Thr 705 710 715 720Glu Leu Arg Lys Val Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val 725 730735 Tyr Lys Gly Ile Trp Ile Pro Asp Gly Glu Asn Val Lys Ile Pro Val 740745 750 Ala Ile Lys Val Leu Arg Glu Asn Thr Ser Pro Lys Ala Asn Lys Glu755 760 765 Ile Leu Asp Glu Ala Tyr Val Met Ala Gly Val Gly Ser Pro TyrVal 770 775 780 Ser Arg Leu Leu Gly Ile Cys Leu Thr Ser Thr Val Gln LeuVal Thr 785 790 795 800 Gln Leu Met Pro Tyr Gly Cys Leu Leu Asp His ValArg Glu His Arg 805 810 815 Gly Arg Leu Gly Ser Gln Asp Leu Leu Asn TrpCys Val Gln Ile Ala 820 825 830 Lys Gly Met Ser Tyr Leu Glu Asp Val ArgLeu Val His Arg Asp Leu 835 840 845 Ala Ala Arg Asn Val Leu Val Lys SerPro Asn His Val Lys Ile Thr 850 855 860 Asp Phe Gly Leu Ala Arg Leu LeuAsp Ile Asp Glu Thr Glu Tyr His 865 870 875 880 Ala Asp Gly Gly Lys ValPro Ile Lys Trp Met Ala Leu Glu Ser Ile 885 890 895 Leu Arg Arg Arg PheThr His Gln Ser Asp Val Trp Ser Tyr Gly Val 900 905 910 Thr Val Trp GluLeu Met Thr Phe Gly Ala Lys Pro Tyr Asp Gly Ile 915 920 925 Pro Ala ArgGlu Ile Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Pro 930 935 940 Gln ProPro Ile Cys Thr Ile Asp Val Tyr Met Ile Met Val Lys Cys 945 950 955 960Trp Met Ile Asp Ser Glu Cys Arg Pro Arg Phe Arg Glu Leu Val Ser 965 970975 Glu Phe Ser Arg Met Ala Arg Asp Pro Gln Arg Phe Val Val Ile Gln 980985 990 Asn Glu Asp Leu Gly Pro Ser Ser Pro Met Asp Ser Thr Phe Tyr Arg995 1000 1005 Ser Leu Leu Glu Asp Asp Asp Met Gly Asp Leu Val Asp AlaGlu 1010 1015 1020 Glu Tyr Leu Val Pro Gln Gln Gly Phe Phe Ser Pro AspPro Thr 1025 1030 1035 Pro Gly Thr Gly Ser Thr Ala His Arg Arg His ArgSer Ser Ser 1040 1045 1050 Thr Arg Ser Gly Gly Gly Glu Leu Thr Leu GlyLeu Glu Pro Ser 1055 1060 1065 Glu Glu Gly Pro Pro Arg Ser Pro Leu AlaPro Ser Glu Gly Ala 1070 1075 1080 Gly Ser Asp Val Phe Asp Gly Asp LeuAla Met Gly Val Thr Lys 1085 1090 1095 Gly Leu Gln Ser Leu Ser Pro HisAsp Leu Ser Pro Leu Gln Arg 1100 1105 1110 Tyr Ser Glu Asp Pro Thr LeuPro Leu Pro Pro Glu Thr Asp Gly 1115 1120 1125 Tyr Val Ala Pro Leu AlaCys Ser Pro Gln Pro Glu Tyr Val Asn 1130 1135 1140 Gln Ser Glu Val GlnPro Gln Pro Pro Leu Thr Pro Glu Gly Pro 1145 1150 1155 Leu Pro Pro ValArg Pro Ala Gly Ala Thr Leu Glu Arg Pro Lys 1160 1165 1170 Thr Leu SerPro Gly Lys Asn Gly Val Val Lys Asp Val Phe Ala 1175 1180 1185 Phe GlyGly Ala Val Glu Asn Pro Glu Tyr Leu Val Pro Arg Glu 1190 1195 1200 GlyThr Ala Ser Pro Pro His Pro Ser Pro Ala Phe Ser Pro Ala 1205 1210 1215Phe Asp Asn Leu Tyr Tyr Trp Asp Gln Asn Ser Ser Glu Gln Gly 1220 12251230 Pro Pro Pro Ser Asn Phe Glu Gly Thr Pro Thr Ala Glu Asn Pro 12351240 1245 Glu Tyr Leu Gly Leu Asp Val Pro Val 1250 1255 3 828 DNA Rat 3atggcctcct gtgtggatct ggatgaacga ggctgcccag cagagcagag agccagcccg 60gtgacattca tcattgcaac tgtagtgggc gtcctgctgt tcctgatctt agtggtggtc 120gttggaatcc taatcaaacg aaggagacag aagatccgga gccctacccc aggcactggg 180agcacagccc atagaaggca ccgcagctcg tccaccagga gtggaggtgg tgagctgaca 240ctgggcctgg agccctcgga agaagggccc cccagatctc cactggctcc ctcggaaggg 300gctggctccg atgtgtttga tggtgacctg gcaatggggg taaccaaagg gctgcagagc 360ctctctccac atgacctcag ccctctacag cggtacagcg aggaccccac attacctctg 420ccccccgaga ctgatggcta tgttgctccc ctggcctgca gcccccagcc cgagtatgtg 480aaccaatcag aggttcagcc tcagcctcct ttaaccccag agggtcctct gcctcctgtc 540cggcctgctg gtgctactct agaaagaccc aagactctct ctcctgggaa gaatggggtt 600gtcaaagacg tttttgcctt cgggggtgct gtggagaacc ctgaatactt agtaccgaga 660gaaggcactg cctctccgcc ccacccttct cctgccttca gcccagcctt tgacaacctc 720tattactggg accagaactc atcggagcag gggcctccac caagtaactt tgaagggacc 780cccactgcag agaaccctga gtacctaggc ctggatgtac ctgtatga 828 4 275 PRT Rat 4Met Ala Ser Cys Val Asp Leu Asp Glu Arg Gly Cys Pro Ala Glu Gln 1 5 1015 Arg Ala Ser Pro Val Thr Phe Ile Ile Ala Thr Val Val Gly Val Leu 20 2530 Leu Phe Leu Ile Leu Val Val Val Val Gly Ile Leu Ile Lys Arg Arg 35 4045 Arg Gln Lys Ile Arg Ser Pro Thr Pro Gly Thr Gly Ser Thr Ala His 50 5560 Arg Arg His Arg Ser Ser Ser Thr Arg Ser Gly Gly Gly Glu Leu Thr 65 7075 80 Leu Gly Leu Glu Pro Ser Glu Glu Gly Pro Pro Arg Ser Pro Leu Ala 8590 95 Pro Ser Glu Gly Ala Gly Ser Asp Val Phe Asp Gly Asp Leu Ala Met100 105 110 Gly Val Thr Lys Gly Leu Gln Ser Leu Ser Pro His Asp Leu SerPro 115 120 125 Leu Gln Arg Tyr Ser Glu Asp Pro Thr Leu Pro Leu Pro ProGlu Thr 130 135 140 Asp Gly Tyr Val Ala Pro Leu Ala Cys Ser Pro Gln ProGlu Tyr Val 145 150 155 160 Asn Gln Ser Glu Val Gln Pro Gln Pro Pro LeuThr Pro Glu Gly Pro 165 170 175 Leu Pro Pro Val Arg Pro Ala Gly Ala ThrLeu Glu Arg Pro Lys Thr 180 185 190 Leu Ser Pro Gly Lys Asn Gly Val ValLys Asp Val Phe Ala Phe Gly 195 200 205 Gly Ala Val Glu Asn Pro Glu TyrLeu Val Pro Arg Glu Gly Thr Ala 210 215 220 Ser Pro Pro His Pro Ser ProAla Phe Ser Pro Ala Phe Asp Asn Leu 225 230 235 240 Tyr Tyr Trp Asp GlnAsn Ser Ser Glu Gln Gly Pro Pro Pro Ser Asn 245 250 255 Phe Glu Gly ThrPro Thr Ala Glu Asn Pro Glu Tyr Leu Gly Leu Asp 260 265 270 Val Pro Val275 5 50 PRT Rat 5 Ser Cys Val Asp Leu Asp Glu Arg Gly Cys Pro Ala GluGln Arg Ala 1 5 10 15 Ser Pro Val Thr Phe Ile Ile Ala Thr Val Val GlyVal Leu Leu Phe 20 25 30 Leu Ile Leu Val Val Val Val Gly Ile Leu Ile LysArg Arg Arg Gln 35 40 45 Lys Ile 50 6 154 DNA Rat 6 tcctgtgtggatctggatga acgaggctgc ccagcagagc agagagccag cccggtgaca 60 ttcatcattgcaactgtaga gggcgtcctg ctgttcctga tcttagtggt ggtcgttgga 120 atcctaatcaaacgaaggag acagaagatc cgga 154 7 221 PRT Rat 7 Pro Thr Pro Gly Thr GlySer Thr Ala His Arg Arg His Arg Ser Ser 1 5 10 15 Ser Thr Arg Ser GlyGly Gly Glu Leu Thr Leu Gly Leu Glu Pro Ser 20 25 30 Glu Glu Gly Pro ProArg Ser Pro Leu Ala Pro Ser Glu Gly Ala Gly 35 40 45 Ser Asp Val Phe AspGly Asp Leu Ala Met Gly Val Thr Lys Gly Leu 50 55 60 Gln Ser Leu Ser ProHis Asp Leu Ser Pro Leu Gln Arg Tyr Ser Glu 65 70 75 80 Asp Pro Thr LeuPro Leu Pro Pro Glu Thr Asp Gly Tyr Val Ala Pro 85 90 95 Leu Ala Cys SerPro Gln Pro Glu Tyr Val Asn Gln Ser Glu Val Gln 100 105 110 Pro Gln ProPro Leu Thr Pro Glu Gly Pro Leu Pro Pro Val Arg Pro 115 120 125 Ala GlyAla Thr Leu Glu Arg Pro Lys Thr Leu Ser Pro Gly Lys Asn 130 135 140 GlyVal Val Lys Asp Val Phe Ala Phe Gly Gly Ala Val Glu Asn Pro 145 150 155160 Glu Tyr Leu Val Pro Arg Glu Gly Thr Ala Ser Pro Pro His Pro Ser 165170 175 Pro Ala Phe Ser Pro Ala Phe Asp Asn Leu Tyr Tyr Trp Asp Gln Asn180 185 190 Ser Ser Glu Gln Gly Pro Pro Pro Ser Asn Phe Glu Gly Thr ProThr 195 200 205 Ala Glu Asn Pro Glu Tyr Leu Gly Leu Asp Val Pro Val 210215 220 8 674 DNA Rat 8 tccggagccc taccccaggc actgggagca cagcccatagaaggcaccgc agctcgtcca 60 ccaggagtgg aggtggtgag ctgacactgg gcctggagccctcggaagaa gggcccccca 120 gatctccact ggctccctcg gaaggggctg gctccgatgtgtttgatggt gacctggcaa 180 tgggggtaac caaagggctg cagagcctct ctccacatgacctcagccct ctacagcggt 240 acagcgagga ccccacatta cctctgcccc ccgagactgatggctatgtt gctcccctgg 300 cctgcagccc ccagcccgag tatgtgaacc aatcagaggttcagcctcag cctcctttaa 360 ccccagaggg tcctctgcct cctgtccggc ctgctggtgctactctagaa agacccaaga 420 ctctctctcc tgggaagaat ggggttgtca aagacgtttttgccttcggg ggtgctgtgg 480 agaaccctga atacttagta ccgagagaag gcactgcctctccgccccac ccttctcctg 540 ccttcagccc agcctttgac aacctctatt actgggaccagaactcatcg gagcaggggc 600 ctccaccaag taactttgaa gggaccccca ctgcagagaaccctgagtac ctaggcctgg 660 atgtacctgt atga 674 9 825 DNA Homo sapiens 9atggcctgtg tggacctgga tgacaagggc tgccccgccg agcagagagc cagccctctg 60acgtccatcg tctctgcggt ggttggcatt ctgctggtcg tggtcttggg ggtggtcttt 120gggatcctca tcaagcgacg gcagcagaag atccggagcc ctgccccggg cgctgggggc 180atggtccacc acaggcaccg cagctcatct accaggagtg gcggtgggga cctgacacta 240gggctggagc cctctgaaga ggaggccccc aggtctccac tggcaccctc cgaaggggct 300ggctccgatg tatttgatgg tgacctggga atgggggcag ccaaggggct gcaaagcctc 360cccacacatg accccagccc tctacagcgg tacagtgagg accccacagt acccctgccc 420tctgagactg atggctacgt tgcccccctg acctgcagcc cccagcctga atatgtgaac 480cagccagatg ttcggcccca gcccccttcg ccccgagagg gccctctgcc tgctgcccga 540cctgctggtg ccactctgga aagggccaag actctctccc cagggaagaa tggggtcgtc 600aaagacgttt ttgcctttgg gggtgccgtg gagaaccccg agtacttgac accccaggga 660ggagctgccc ctcagcccca ccctcctcct gccttcagcc cagccttcga caacctctat 720tactgggacc aggacccacc agagcggggg gctccaccca gcaccttcaa agggacacct 780acggcagaga acccagagta cctgggtctg gacgtgccag tgtga 825 10 275 PRT Homosapiens 10 Met Ala Cys Val Asp Leu Asp Asp Lys Gly Cys Pro Ala Glu GlnArg 1 5 10 15 Ala Ser Pro Leu Thr Ser Ile Val Ser Ala Val Val Gly IleLeu Leu 20 25 30 Val Val Val Leu Gly Val Val Phe Gly Ile Leu Ile Lys ArgArg Gln 35 40 45 Gln Lys Ile Arg Ser Pro Ala Pro Gly Ala Gly Gly Met ValHis His 50 55 60 Arg His Arg Ser Ser Ser Thr Arg Ser Gly Gly Gly Asp LeuThr Leu 65 70 75 80 Gly Leu Glu Pro Ser Glu Glu Glu Ala Pro Arg Ser ProLeu Ala Pro 85 90 95 Ser Glu Gly Ala Gly Ser Asp Val Phe Asp Gly Asp LeuGly Met Gly 100 105 110 Ala Ala Lys Gly Leu Gln Ser Leu Pro Thr His AspPro Ser Pro Leu 115 120 125 Gln Arg Tyr Ser Glu Asp Pro Thr Val Pro LeuPro Ser Glu Thr Asp 130 135 140 Gly Tyr Val Ala Pro Leu Thr Cys Ser ProGln Pro Glu Tyr Val Asn 145 150 155 160 Gln Pro Asp Val Arg Pro Gln ProPro Ser Pro Arg Glu Gly Pro Leu 165 170 175 Pro Ala Ala Arg Pro Ala GlyAla Thr Leu Glu Arg Ala Lys Thr Leu 180 185 190 Ser Pro Gly Lys Asn GlyVal Val Lys Asp Val Phe Ala Phe Gly Gly 195 200 205 Ala Val Glu Asn ProGlu Tyr Leu Thr Pro Gln Gly Gly Ala Ala Pro 210 215 220 Gln Pro His ProPro Pro Ala Phe Ser Pro Ala Phe Asp Asn Leu Tyr 225 230 235 240 Tyr TrpAsp Gln Asp Pro Pro Glu Arg Gly Ala Pro Pro Ser Thr Phe 245 250 255 LysGly Thr Pro Thr Ala Glu Asn Pro Glu Tyr Leu Gly Leu Asp Val 260 265 270Pro Val Glx 275 11 50 PRT Homo sapiens 11 Cys Val Asp Leu Asp Asp LysGly Cys Pro Ala Glu Gln Arg Ala Ser 1 5 10 15 Pro Leu Thr Ser Ile IleSer Ala Val Val Gly Ile Leu Leu Val Val 20 25 30 Val Leu Gly Val Val PheGly Ile Leu Ile Lys Arg Arg Gln Gln Lys 35 40 45 Ile Arg 50 12 151 DNAHomo sapiens 12 tgtgtggacc tggatgacaa gggctgcccc gccgagcaga gagccagccctctgacgtcc 60 atcgtctctg cggtggttgg cattctgctg gtcgtggtct tgggggtggtctttgggatc 120 ctcatcaagc gacggcagca gaagatccgg a 151 13 224 PRT Homosapiens 13 Cys Pro Asp Pro Ala Pro Gly Ala Gly Gly Met Val His His ArgHis 1 5 10 15 Arg Ser Ser Ser Thr Arg Ser Gly Gly Gly Asp Leu Thr LeuGly Leu 20 25 30 Glu Pro Ser Glu Glu Glu Ala Pro Arg Ser Pro Leu Ala ProSer Glu 35 40 45 Gly Ala Gly Ser Asp Val Phe Asp Gly Asp Leu Gly Met GlyAla Ala 50 55 60 Lys Gly Leu Gln Ser Leu Pro Thr His Asp Pro Ser Pro LeuGln Arg 65 70 75 80 Tyr Ser Glu Asp Pro Thr Val Pro Leu Pro Ser Glu ThrAsp Gly Tyr 85 90 95 Val Ala Pro Leu Thr Cys Ser Pro Gln Pro Glu Tyr ValAsn Gln Pro 100 105 110 Asp Val Arg Pro Gln Pro Pro Ser Pro Arg Glu GlyPro Leu Pro Ala 115 120 125 Ala Arg Pro Ala Gly Ala Thr Leu Glu Arg ProLys Thr Leu Ser Pro 130 135 140 Gly Lys Asn Gly Val Val Lys Asp Val PheAla Phe Gly Gly Ala Val 145 150 155 160 Glu Asn Pro Glu Tyr Leu Thr ProGln Gly Gly Ala Ala Pro Gln Pro 165 170 175 His Pro Pro Pro Ala Phe SerPro Ala Phe Asp Asn Leu Tyr Tyr Trp 180 185 190 Asp Gln Asp Pro Pro GluArg Gly Ala Pro Pro Ser Thr Phe Lys Gly 195 200 205 Thr Pro Thr Ala GluAsn Pro Glu Tyr Leu Gly Leu Asp Val Pro Val 210 215 220 14 679 DNA Homosapiens misc_feature (9)..(9) n represents a site specific a to gmutation induced to create the BspE1 site for fusion 14 ttctgtccngagccctgccc cgggcgctgg gggcatggtc caccacaggc accgcagctc 60 atctaccaggagtggcggtg gggacctgac actagggctg gagccctctg aagaggaggc 120 ccccaggtctccactggcac cctccgaagg ggctggctcc gatgtatttg atggtgacct 180 gggaatgggggcagccaagg ggctgcaaag cctccccaca catgacccca gccctctaca 240 gcggtacagtgaggacccca cagtacccct gccctctgag actgatggct acgttgcccc 300 cctgacctgcagcccccagc ctgaatatgt gaaccagcca gatgttcggc cccagccccc 360 ttcgccccgagagggccctc tgcctgctgc ccgacctgct ggtgccactc tggaaagggc 420 caagactctctccccaggga agaatggggt cgtcaaagac gtttttgcct ttgggggtgc 480 cgtggagaaccccgagtact tgacacccca gggaggagct gcccctcagc cccaccctcc 540 tcctgccttcagcccagcct tcgacaacct ctattactgg gaccaggacc caccagagcg 600 gggggctccacccagcacct tcaaagggac acctacggca gagaacccag agtacctggg 660 tctggacgtgccagtgtga 679

1. A recombinant polynucleotide, comprising a first nucleotide sequenceencoding a Her2/neu target antigen consisting of an amino acid sequencecorresponding to about amino acid residues 634 to 683 operatively linkedto amino acid residues 1035 to 1255 of SEQ ID NO:1, or amino acidresidues 635 to 685 operatively linked to amino acid residues 1037 to1257 of SEQ ID NO:2.
 2. The recombinant polynucleotide of claim 1,wherein the Her2/neu target antigen has an amino acid sequence as setforth in SEQ ID NO:4 or SEQ ID NO:9.
 3. The recombinant polynucleotideof claim 2, which has a first nucleotide sequence as set forth in SEQ IDNO:4 or SEQ ID NO:10.
 4. The recombinant polynucleotide of claim 1,further comprising a second nucleotide sequence, which is operativelylinked to the first nucleotide sequence.
 5. The recombinantpolynucleotide of claim 4, wherein the second nucleotide sequencecomprises an expression regulatory element.
 6. The recombinantpolynucleotide of claim 5, wherein the expression regulatory element isa transcriptional regulatory element, a translational regulatoryelement, or a combination thereof.
 7. The recombinant polynucleotide ofclaim 4, wherein the second nucleotide sequence encodes a heterologousamino acid sequence.
 8. The recombinant polynucleotide of claim 7, whichencodes a fusion protein comprising the Her2/neu target antigen and theheterologous amino acid sequence.
 9. The recombinant polynucleotide ofclaim 7, wherein the heterologous amino acid sequence is a polypeptide.10. The recombinant polynucleotide of claim 9, wherein the polypeptideis an immunostimulatory polypeptide.
 11. A cell containing thepolynucleotide of claim
 1. 12. The cell of claim 11, which is amammalian cell.
 13. A recombinant polynucleotide, comprising a firstnucleotide sequence encoding a Her2/neu target antigen consisting of anamino acid sequence corresponding to amino acid residues 606 to 683operatively linked to amino acid residues 1035 to 1255 of SEQ ID NO:1,or amino acid residues 608 to 685 operatively linked to amino acidresidues 1037 to 1257 of SEQ ID NO:2.
 14. A vector, comprising therecombinant polynucleotide of claim 1 or claim
 13. 15. The vector ofclaim 14, which is an expression vector.
 16. The vector of claim 15,wherein the expression vector is a mammalian cell expression vector. 17.The vector of claim 14, which is a viral vector.
 18. The vector of claim17, wherein the viral vector is an alphavirus vector.
 19. The vector ofclaim 18, wherein the alphavirus vector is a Venezuelan equineencephalitis virus (VEE) vector.
 20. The vector of claim 17, wherein theviral vector is a retrovirus vector, an adenovirus vector, anadeno-associated virus vector, or a vaccinia virus vector.
 21. Thevector of claim 14, which is a plasmid vector.
 22. The vector of claim21, wherein the plasmid vector is a viral vector plasmid.
 23. A hostcell containing the vector of claim
 14. 24-28. (Cancelled)
 29. Arecombinant polypeptide, comprising a Her2/neu target antigen consistingof an amino acid sequence corresponding to about amino acid residues 634to 683 operatively linked to amino acid residues 1035 to 1255 of SEQ IDNO:1, or amino acid residues 635 to 685 operatively linked to amino acidresidues 1037 to 1257 of SEQ ID NO:2.
 30. The recombinant polypeptide ofclaim 29, wherein the Her2/neu target antigen has an amino acid sequenceset forth in SEQ ID NO:4 or SEQ ID NO:10, or an amino acid sequencecorresponding thereto. 31-32. (Cancelled)
 33. A recombinant polypeptide,comprising a Her2/neu target antigen consisting of an amino acidsequence corresponding to amino acid residues 606 to 683 operativelylinked to amino acid residues 1035 to 1255 of SEQ ID NO:1, or amino acidresidues 635 to 685 operatively linked to amino acid residues 1037 to1257 of SEQ ID NO:2. 34-74. (Cancelled)